Long-term Proliferation Research Articles

Evaluation of enzymatic protocols to optimize efficiency of bovine adipose tissue-derived mesenchymal stromal cell isolation

Sustainable food provision for a continuously growing human population is one of the major challenges for the next decades. Cultured meat represents one of the alternatives which is currently extensively explored. Yet, the most appropriate cell type, capable of long-term proliferation and myogenic differentiation, remains to be identified. Bovine mesenchymal stromal cells (MSCs) are considered as a promising cell source. Within the context of cultured meat production, it is mandatory to maximize cell yield per tissue source. Although many enzymatic methods to isolate MSCs from adipose tissue (AT) have been described, cell yield has never been compared. In this study, we evaluate 32 isolation conditions including four enzyme mixtures (Collagenase type I, Collagenase type I + Trypsin, LiberaseTM and Collagenase type IV) at varying concentrations and incubation times, regarding their efficiency to isolate MSCs from bovine subcutaneous AT. The highest cell yield in combination with a low population doubling time was obtained using LiberaseTM at a concentration of 0.1% for 3 h. MSC identity of the cells was confirmed by tri-lineage differentiation potential and cell surface marker expression. Subsequently, isolated cells were myogenically differentiated using 5-aza-2’-deoxycytidine and galectin-1. mRNA levels of the myogenic regulatory factors (MRF) myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), MYF6, and myogenin (MYOG) were increased, while less paired box 3 (PAX3) mRNA expression was observed when compared to undifferentiated MSCs. The presence of desmin (DES), tropomyosin (TM), and myosin heavy chain (MyHC) in myogenically differentiated bovine AT-MSCs was confirmed using immunofluorescence stainings. When considering MSCs from bovine AT as potential cell source to produce cultured meat, it is recommended to use 0.1% LiberaseTM for 3 h to ensure a high cell yield.

Antibacterial and cytocompatible silver coating for titanium Boston Keratoprosthesis.

The Boston Keratoprosthesis (BKPro) serves as a medical solution for restoring vision in complex cases of corneal blindness. Comprising a front plate made of polymethylmethacrylate (PMMA) and a back plate of titanium (Ti), this device utilizes the beneficial biomaterial properties of Ti. While BKPro demonstrates promising retention rates, infection emerges as a significant concern that impacts its long-term efficacy. However, limited research exists on enhancement of BKPros through intrinsic infection-preventing mechanisms. In this regard, metal ions, especially the well-known Ag+ ions, are a promising alternative to obtain implants with innate antibacterial properties. However, little information is available about the effects of Ag in corneal tissue, especially within human corneal keratocytes (HCKs). In this work, an electrodeposition treatment using a constant pulse is proposed to attach Ag complexes onto rough Ti surfaces, thus providing antibacterial properties without inducing cytotoxicity. Complete physicochemical characterization and ion release studies were carried out with both control and Ag-treated samples. The possible cytotoxic effects in the short and long term were evaluated in vitro with HCKs. Moreover, the antibacterial properties of the silver-treated surfaces were tested against the gram-negative bacterial strain Pseudomonas aeruginosa and the gram-positive strain Staphylococcus epidermidis, that are common contributors to infections in BKPros. Physicochemical characterization confirmed the presence of silver, predominantly in oxide form, with low release of Ag+ ions. Ag-treated surfaces demonstrated no cytotoxicity and promoted long-term proliferation of HCKs. Furthermore, the silver-treated surfaces exhibited a potent antibacterial effect, causing a reduction in bacterial adhesion and evident damage to the bacterial cell walls of P. aeruginosa and S. epidermidis. The low release of Ag+ ions suggested reactive oxygen species (ROS)-mediated oxidative stress imbalance as the bactericidal mechanism of the silver deposits. In conclusion, the proposed electrodeposition technique confers antibacterial protection to the Ti backplate of BKPro, mitigating implant-threatening infections while ensuring non-cytotoxicity within the corneal tissue.

Further biological characterization of small molecules UM171 and SR1: In vitro effects on three hematopoietic cell populations from human cord blood

Small molecules UM171 and SR1 have already been taken into clinically-oriented protocols for the ex vivo expansion of hematopoietic stem (HSCs) and progenitor (HPCs) cells. In order to gain further insight into their biology, in the present study we have assessed their effects, both individually and in combination, on the in vitro long-term proliferation and expansion of HSCs and HPCs contained within three different cord blood-derived cell populations: MNCs (CD34+ cells = 0.8 %), LIN− cells (CD34+ cells = 41 %), and CD34+ cells (CD34+ cells >98 %). Our results show that when added to cultures in the absence of recombinant stimulatory cytokines, neither molecule had any effect. In contrast, when added in the presence of hematopoietic cytokines, UM171 and SR1 had significant stimulatory effects on cell proliferation and expansion in cultures of LIN− and CD34+ cells. No significant effects were observed in cultures of MNCs. The effects of both molecules were more pronounced in cultures with the highest proportion of CD34+ cells, and the greatest effects were observed when both molecules were added in combination. In the absence of small molecules, cell numbers reached a peak by days 25–30, and then declined; whereas in the presence of UM171 or/and SR1 cell numbers were sustained up to day 45 of culture. Our results indicate that besides CD34+ cells, LIN− cells could also be used as input cells in clinically-oriented expansion protocols, and that using both molecules simultaneously would be a better approach than using only one of them.

Triamcinolone acetonide has minimal effect on short- and long-term metabolic activities of cartilage.

Intra-articular corticosteroid injections, such as triamcinolone acetonide (TA), are commonly used by clinicians to manage joint synovial inflammation. However, due to conflicting evidence in literature, there is a fear among clinicians that the injections may be harmful to otherwise healthy cartilage in young patients. The purpose of this study was to evaluate the effects of TA on young, healthy chondrocytes. Articular cartilage samples were harvested from bovine knee joints (1-2 months old). In both healthy and inflammatory (interleukin-1β) challenged cartilage, samples were treated with TA at doses ranging from 1 nM to 200 μM. Following a short- (2 days) or long-term (10-14 days) treatment, chondrocyte viability, proliferation, and extracellular matrix (ECM) synthesis and degradation were evaluated with a click chemistry-based technique. Chondrocyte viability, proliferation, and anabolic activity were all minimally affected by short-term and long-term TA treatment. After both acute and sustained inflammatory challenges, TA reduced the catabolic activities in cartilage, reducing nascent glycosaminoglycan loss and maintaining cartilage mechanical properties. Overall, at physiologically relevant doses, TA had minimal negative impact on chondrocytes when maintained within their native ECM.Clinical significance: The findings provide new insight for current clinical practices concerning the use of TA in intra-articular injections, especially in young patients, and established a foundation for future investigations into the impact of corticosteroids on joint homeostasis.

BCG priming followed by a novel interleukin combination activates Natural Killer cells to selectively proliferate and become anti-tumour long-lived effectors

The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients’ bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.

Presenting dual-functional peptides on implant surface to direct in vitro osteogenesis and in vivo osteointegration

The complex biological process of osseointegration and the bio-inertness of bone implants are the major reasons for the high failure rate of long-term implants, and have also promoted the rapid development of multifunctional implant coatings in recent years. Herein, through the special design of peptides, we use layer-by-layer assembly technology to simultaneously display two peptides with different biological functions on the implant surface to address this issue. A variety of surface characterization techniques (ellipsometry, atomic force microscopy, photoelectron spectroscopy, dissipation-quartz crystal microbalance) were used to study in detail the preparation process of the dual peptide functional coating and the physical and chemical properties, such as the composition, mechanical modulus, stability, and roughness of the coating. Compared with single peptide functional coatings, dual-peptide functionalized coatings had much better performances on antioxidant, cellular adhesion in early stage, proliferation and osteogenic differentiation in long term, as well as in vivo osteogenesis and osseointegration capabilities. These findings will promote the development of multifunctional designs in bone implant coatings, as a coping strategy for the complexity of biological process during osteointegration.

A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.

Comparative study of cell interaction and bacterial adhesion on titanium of different composition, structure and surfaces with various laser treatment

Abstract Titanium and its alloys are commonly used in modern implantology. Cell viability on the surface of titanium implants depends on the surface topography, roughness, and wettability. Laser treatment is a successful method to control the surface morphology.
The aim of this study was to comprehensively investigate the effects of laser ablation on titanium surfaces and their interactions with cells and bacteria. Cell adhesion, proliferation, and bacterial retention on smooth and laser-textured samples of commercially pure and nanostructured titanium of two grades were evaluated. Femtosecond laser treatment effectively enhances the wettability. Titanium grade four exhibits superior adhesion and proliferation rates when compared to titanium grade two. The cytotoxicity of nanostructured titanium is significantly lower, regardless of the surface treatment. Laser treatment resulted in increased short-term cell proliferation on grade two titanium and long-term cell proliferation on nanostructured grade two titanium only. Although the laser ablation has a limited effect on bacterial adhesion, the coverage of less than 1% in most samples indicates that the material itself has an antibacterial effect on the bacterial strain S. oralis.
These findings provide valuable insights into how different material structures and surface treatments can affect cellular response and antibacterial properties for potential use in dental implantology.

Bone-like microtextures of HA coatings prepared by nanosecond laser and their properties

Although the hydroxyapatite (HA) coating implant prepared by laser cladding exhibits good biological activity due to its chemical composition similar to biological bone, the original surface roughness of the laser-clad coating is high, which is unfavorable for long-term cell adhesion and proliferation. In order to improve the biological activity of HA coating implants and increase the adhesion and proliferation rate of osteoblasts on their surface, microtextures with different length-diameter ratios and depth gradients were prepared on the HA coating surface by nanosecond laser with reference to the microscopic morphology characteristics of the biological bone surface. By investigating the effects of microtexture aspect ratio and depth gradient on osteoblast adhesion characteristics, it was found that bone-like microtextures better controlled and simulated cell-material surface interactions. The geometric morphology of microtextured bone with a wide front end and narrow rear end and a depth gradient from front to back was an important signal for osteoblast proliferation. When the aspect ratio of bone microtexture was 2.5:1 and the maximum depth gradient was 15.7 μm, osteoblasts within the microtexture showed optimal growth, proliferation, and adhesion. Additionally, due to induction by microtexture contour shape and depth gradient morphology, cell morphology showed varying adhesion morphology from the front to the back of the microtexture. The results demonstrated that HA coatings with bone-like microtextures had superior osteoblast proliferation and adhesion properties compared to the original cladded coating surface, beneficial for enhancing the application of HA-coated bone implants in large segment bone repair.

Abstract 1101: A patient-derived xenograft in vitro platform identifies panobinostat as the most efficient drug to treat high-risk hepatoblastoma patients

Abstract Hepatoblastoma is the most common pediatric liver tumor, and the treatment of patients with preoperative chemotherapy and surgical resection of the tumor has led to survival rates of up to 83%. However, resistance of the primary tumor towards chemotherapy and the persistence of metastases still represents major challenges to achieve complete remission in high-risk patients, which is associated with poor prognosis. In order to identify new treatment options, we have used a newly developed in vitro drug-testing platform comprised of conventional cell lines and cultures from dissociated patient-derived xenografts of pediatric liver cancers to test standard-of-care medications used in the current PHITT trial as well as compounds targeting a wide range of different cellular pathways. We identified histone deacetylase (HDAC) inhibitors as a promising drug family to reduce tumor cell growth in a dose-dependent manner, with panobinostat being the most effective drug tested. Subsequent in vitro assays showed that panobinostat reduced short- and long-term proliferation, retarded three-dimensional spheroid growth and ultimately induced apoptosis of hepatoblastoma cells. RNA sequencing indicated that panobinostat results in downregulation of MYC target genes and upregulation of the dual specificity phosphatase 1. Of clinical relevance, the combination of panobinostat with doxorubicin revealed a strong synergistic effect, even stronger than the one of cisplatin and doxorubicin, which is currently given to high-risk hepatoblastoma patients. Moreover, increased expression of HDAC4 and HDAC11 in primary tumor tissues was significantly associated with metastatic disease. Our results strongly suggest panobinostat to be used in future therapeutic trials for high-risk hepatoblastoma patients, especially when combined with doxorubicin. Citation Format: Salih Demir, Emilie Indersie, Sophie Branchereau, Eiso Hiyama, Stefano Cairo, Roland Kappler. A patient-derived xenograft in vitro platform identifies panobinostat as the most efficient drug to treat high-risk hepatoblastoma patients [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 1101.

Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors.

Previously, we found that Wnt and Notch signaling govern stem cells of clear cell kidney cancer (ccRCC) in patients. To mimic stem cell responses in the normal kidney in vitro in a marker-unbiased fashion, we have established tubular organoids (tubuloids) from total single adult mouse kidney epithelial cells in Matrigel and serum-free conditions. Deep proteomic and phosphoproteomic analyses revealed that tubuloids resembled renewal of adult kidney tubular epithelia, since tubuloid cells displayed activity of Wnt and Notch signaling, long-term proliferation and expression of markers of proximal and distal nephron lineages. In our wish to model stem cell-derived human ccRCC, we have generated two types of genetic double kidney mutants in mice: Wnt-β-catenin-GOF together with Notch-GOF and Wnt-β-catenin-GOF together with a most common alteration in ccRCC, Vhl-LOF. An inducible Pax8-rtTA-LC1-Cre was used to drive recombination specifically in adult kidney epithelial cells. We confirmed mutagenesis of β-catenin, Notch and Vhl alleles on DNA, protein and mRNA target gene levels. Surprisingly, we observed symptoms of chronic kidney disease (CKD) in mutant mice, but no increased proliferation and tumorigenesis. Thus, the responses of kidney stem cells in the tubuloid and genetic systems produced different phenotypes, i.e. enhanced renewal versus CKD.

RhIL-7-hyFc and hIL-2/TCB2c combination promotes an immune-stimulatory tumor microenvironment that improves antitumor efficacy of checkpoint inhibitors

BackgroundRecombinant human interleukin (rhIL)-7-hyFc (efineptakin alfa; NT-I7) is a potent T-cell amplifier, with two IL-7 molecules fused to IgD/IgG4 elements. rhIL-7-hyFc promotes extensive infiltration of CD8+ T cells into the...

Preparation and in vitro evaluation of cell adhesion and long-term proliferation of stem cells cultured on silibinin co-embedded PLGA/Collagen electrospun composite nanofibers

The present research aims to evaluate the efficacy of Silibinin-loaded mesoporous silica nanoparticles (Sil@MSNs) immobilized into polylactic-co-glycolic acid/Collagen (PLGA/Col) nanofibers on the in vitro proliferation of adipose-derived stem cells (ASCs) and cellular senescence. Here, the fabricated electrospun PLGA/Col composite scaffolds were coated with Sil@MSNs and their physicochemical properties were examined by FTIR, FE-SEM, and TGA. The growth, viability and proliferation of ASCs were investigated using various biological assays including PicoGreen, MTT, and RT-PCR after 21 days. The proliferation and adhesion of ASCs were supported by the biological and mechanical characteristics of the Sil@MSNs PLGA/Col composite scaffolds, according to FE- SEM. PicoGreen and cytotoxicity analysis showed an increase in the rate of proliferation and metabolic activity of hADSCs after 14 and 21 days, confirming the initial and controlled release of Sil from nanofibers. Gene expression analysis further confirmed the increased expression of stemness markers as well as hTERT and telomerase in ASCs seeded on Sil@MSNs PLGA/Col nanofibers compared to the control group. Ultimately, the findings of the present study introduced Sil@MSNs PLGA/Col composite scaffolds as an efficient platform for long-term proliferation of ASCs in tissue engineering.

Organoid co-culture models of the tumor microenvironment promote precision medicine.

In recent years, the three-dimensional(3D) culture system has emerged as a promising preclinical model for tumor research owing to its ability to replicate the tissue structure and molecular characteristics of solid tumors in vivo. This system offers several advantages, including high throughput, efficiency, and retention of tumor heterogeneity. Traditional Matrigel-submerged organoid cultures primarily support the long-term proliferation of epithelial cells. One solution for the exploration of the tumor microenvironment is a reconstitution approach involving the introduction of exogenous cell types, either in dual, triple or even multiple combinations. Another solution is a holistic approach including patient-derived tumor fragments, air-liquid interface, suspension 3Dculture, and microfluidic tumor-on-chip models. Organoid co-culture models have also gained popularity for studying the tumor microenvironment, evaluating tumor immunotherapy, identifying predictive biomarkers, screening for effective drugs, and modeling infections. By leveraging these 3Dculture systems, it is hoped to advance the clinical application of therapeutic approaches and improve patient outcomes.

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Targeting Endothelial PERK-DLL4 Axis to Enhance Hematopoietic Stem Cell and Lymphoid Progenitor Regeneration

Despite our current information on hundreds of genes implicated in hematopoietic stem cells (HSC) biology, major gaps still exist in our understanding of HSC physiology and their regeneration mechanism under disease and stress conditions, hindering the development of strategies to enhance HSC regeneration. HSCs reside in a tightly controlled bone marrow HSC niche that regulates and maintains hematopoietic homeostasis. HSC niche is composed of a heterogeneous population of specialized stroma cells. Among these, the endothelial cells and the perivascular stroma cells provide critical pro-hematopoietic factors to support HSC pool and committed lineage progenitors particularly lymphoid progenitors. In addition, evidence suggests that endothelium-expressing Notch ligand may promote Notch-dependent long-term HSC proliferation. Recent single cell transcriptome profiling revealed that Delta-like 4 (DLL4) is expressed by the vascular endothelial cells at a higher level than other Notch ligands while its expression down-regulates in response to myeloablation. However, the molecular mechanism regulating the dynamic DLL4 expression during hematopoietic regeneration in response to myeloablative stimuli is unknown. Unfolded protein response (UPR) (also known as endoplasmic reticulum stress, ER stress) has been increasingly recognized as crucial for HSC maintenance. During hematopoietic regeneration, UPR is critical for meeting the increased demand for protein synthesis required for rapid cellular proliferation and for adapting to pathological conditions such as increased levels of reactive oxygen species following irradiation or chemotherapy. Unlike HSCs, how HSC niche endothelial cells deal with redox imbalance and higher requirements of vessel regeneration to regulate stress hematopoietic regeneration is not clear. In this study, using genetically engineered mouse models, we examined the dynamic expression and function of PERK, one arm of UPR, and its potential downstream target, Dll4, during the stress hematopoiesis triggered by transplantation. We found that PERK is activated in the mouse bone marrow endothelial cells following irradiation. Ablation of endothelial Perk in mice (referred as Perk iΔEC mice) promoted post-transplantation immediate recovery of blood cells derived from wild type donor mice and enhanced HSC and lymphoid progenitor regeneration with the expansion of B lineage progenitors including pre-proB, pro-B, and pre-B cells. In addition, by whole-mount immunostaining and intravital microscopy, we found that endothelial Perk deletion restrained disorganized angiogenesis and vascular leakage provoked by irradiation. Although irradiation itself had no effect on endothelial Dll4, Perk ablation markedly increased endothelial Dll4 expression. We found that Dll4 is essential for the post-irradiation HSC and lymphoid progenitor rejuvenation as ablation of endothelial Dll4 in mice (referred as Dll4 iΔEC mice) led to impaired short-term and long-term hematopoietic regeneration and markedly decreased wild type donor derived peripheral B cell and lymphoid progenitor recovery. We found that endothelial Dll4 is required for HSC quiescence maintenance, HSC niche retention and HSC self-renewal. Further, ablation of endothelial Dll4 led to disorganized bone marrow angiogenesis and increased marrow vascular leakage. Finally, we showed that deletion of Dll4 in Perk iΔEC mice completely abrogated the increased numbers of HSC and lymphoid progenitors mediated by Perk deletion, suggesting that up-regulation of endothelial Dll4 is likely responsible for Perk ablation-mediated enhanced HSC and lymphoid progenitor regeneration. Taken together, our results revealed a novel regulatory mechanism by which endothelial Dll4 expression is suppressed by PERK of UPR during transplantation while ablating Perk increases Dll4 expression. Endothelial DLL4 in the bone marrow endothelial cells is critical for supporting vascular integrity and myeloablation-induced HSC and lymphoid progenitor regeneration. Our findings suggest that targeting endothelial PERK-DLL4 axis could be a viable option to improve post-irradiation regeneration of HSC and lymphoid progenitors.

Optimizing PCL/PLGA Scaffold Biocompatibility Using Gelatin from Bovine, Porcine, and Fish Origin.

This research introduces a novel approach by incorporating various types of gelatins, including bovine, porcine, and fish skin, into polycaprolactone and poly (lactic-co-glycolic acid) using a solvent casting method. The films are evaluated for morphology, mechanical properties, thermal stability, biodegradability, hemocompatibility, cell adhesion, proliferation, and cytotoxicity. The results show that the incorporation of gelatins into the films alters their mechanical properties, with a decrease in tensile strength but an increase in elongation at break. This indicates that the films become more flexible with the addition of gelatin. Gelatin incorporation has a limited effect on the thermal stability of the films. The composites with the gelatin show higher biodegradability with the highest weight loss in the case of fish gelatin. The films exhibit high hemocompatibility with minimal hemolysis observed. The gelatin has a dynamic effect on cell behavior and promotes long-term cell proliferation. In addition, all composite films reveal exceptionally low levels of cytotoxicity. The combination of the evaluated parameters shows the appropriate level of biocompatibility for gelatin-based samples. These findings provide valuable insights for future studies involving gelatin incorporation in tissue engineering applications.

Ergolide mediates anti-cancer effects on metastatic uveal melanoma cells and modulates their cellular and extracellular vesicle proteomes.

Uveal melanoma is a poor prognosis cancer. Ergolide, a sesquiterpene lactone isolated from Inula Brittanica, exerts anti-cancer properties. The objective of this study was to 1) evaluate whether ergolide reduced metastatic uveal melanoma (MUM) cell survival/viability in vitro and in vivo; and 2) to understand the molecular mechanism of ergolide action. Ergolide bioactivity was screened via long-term proliferation assay in UM/MUM cells and in zebrafish MUM xenograft models. Mass spectrometry profiled proteins modulated by ergolide within whole cell or extracellular vesicle (EVs) lysates of the OMM2.5 MUM cell line. Protein expression was analyzed by immunoblots and correlation analyses to UM patient survival used The Cancer Genome Atlas (TCGA) data. Ergolide treatment resulted in significant, dose-dependent reductions (48.5 to 99.9%; p<0.0001) in OMM2.5 cell survival in vitro and of normalized primary zebrafish xenograft fluorescence (56%; p<0.0001) in vivo, compared to vehicle controls. Proteome-profiling of ergolide-treated OMM2.5 cells, identified 5023 proteins, with 52 and 55 proteins significantly altered at 4 and 24 hours, respectively ( p<0.05; fold-change >1.2). Immunoblotting of heme oxygenase 1 (HMOX1) and growth/differentiation factor 15 (GDF15) corroborated the proteomic data. Additional proteomics of EVs isolated from OMM2.5 cells treated with ergolide, detected 2931 proteins. There was a large overlap with EV proteins annotated within the Vesiclepedia compendium. Within the differentially expressed proteins, the proteasomal pathway was primarily altered. Interestingly, BRCA2 and CDKN1A Interacting Protein (BCCIP) and Chitinase Domain Containing 1 (CHID1), were the only proteins significantly differentially expressed by ergolide in both the OMM2.5 cellular and EV isolates and they displayed inverse differential expression in the cells versus the EVs. Ergolide is a novel, promising anti-proliferative agent for UM/MUM. Proteomic profiling of OMM2.5 cellular/EV lysates identified candidate pathways elucidating the action of ergolide and putative biomarkers of UM, that require further examination.

Adipose-Derived Mesenchymal Stem Cell Facilitate Hematopoietic Stem Cell Proliferation via the Jagged-1/Notch-1/Hes Signaling Pathway.

Poor graft function (PGF) is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Current therapies, such as CD34+ cell infusion, have shown limited effectiveness. Conversely, mesenchymal stem cells (MSCs) show potential in addressing PGF. Adipose-derived mesenchymal stem cells (ADSCs) effectively support long-term hematopoietic stem cell proliferation. Therefore, this study aimed to investigate the mechanisms underlying the long-term hematopoietic support provided by ADSCs. ADSCs were isolated from mice and subsequently identified. In vitro experiments involved coculturing ADSCs as feeders with Lin-Sca-1+c-kit+ (LSK) cells from mice for 2 and 5 weeks. The number of LSK cells was quantified after coculture. Scanning electron microscopy was utilized to observe the interaction between ADSCs and LSK cells. Hes-1 expression was assessed using western blot and real-time quantitative PCR. An γ-secretase inhibitor (GSI) was used to confirm the involvement of the Jagged-1/Notch-1/Hes-1 pathway in LSK cell expansion. Additionally, Jagged-1 was knocked down in ADSCs to demonstrate its significance in ADSC-mediated hematopoietic support. In vivo experiments were conducted to study the hematopoietic support provided by ADSCs through the infusion of LSK, LSK + fibroblasts, and LSK + ADSCs, respectively. Mouse survival, platelet count, leukocyte count, and hemoglobin levels were monitored. ADSCs showed high-Jagged-1 expression and promoted LSK cell proliferation. There was a direct interaction between ADSCs and LSK cells. After coculture, Hes-1 expression increased in LSK cells. Moreover, GSI-reduced LSK cell proliferation and Hes-1 expression. Knockdown of Jagged-1 attenuated ADSCs-mediated promotion of LSK cell proliferation. Furthermore, ADSCs facilitated hematopoietic recovery and promoted the survival of NOD/SCID mice. The hematopoietic support provided by ADSCs both in vivo and in vitro may be mediated, at least in part, through the Jagged-1/Notch-1 signaling pathway. These findings provide valuable insights into the mechanisms underlying ADSCs-mediated hematopoietic support and may have implications for improving the treatment of PGF following HSCT.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.