Size-Based Microfluidic-Enriched Mesenchymal Stem Cell Subpopulations Enhance Articular Cartilage Repair.

BACKGROUND: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing. HYPOTHESIS: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair. STUDY DESIGN: Controlled laboratory study. METHODS: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro-computed tomography, and mechanical analysis. RESULTS: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro-computed tomography of the subchondral bone. CONCLUSION: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs. CLINICAL RELEVANCE: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes.

Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties.

BACKGROUND: Mesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD. METHODS: Human bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination. RESULTS: As compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls. CONCLUSION: Our study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Emerging Preclinical Applications of Humanized Mouse Models in the Discovery and Validation of Novel Immunotherapeutics and Their Mechanisms of Action for Improved Cancer Treatment.

Cancer therapeutics have undergone immense research over the past decade. While chemotherapies remain the mainstay treatments for many cancers, the advent of new molecular techniques has opened doors for more targeted modalities towards cancer cells. Although immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy in treating cancer, adverse side effects related to excessive inflammation are often reported. There is a lack of clinically relevant animal models to probe the human immune response towards ICI-based interventions. Humanized mouse models have emerged as valuable tools for pre-clinical research to evaluate the efficacy and safety of immunotherapy. This review focuses on the establishment of humanized mouse models, highlighting the challenges and recent advances in these models for targeted drug discovery and the validation of therapeutic strategies in cancer treatment. Furthermore, the potential of these models in the process of uncovering novel disease mechanisms is discussed.

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles.

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1ß-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs' size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.

Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.

Detection of cellular senescence is important quality analytics of cell therapy products, including mesenchymal stromal cells (MSCs). However, its detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells in heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry, which is related to intracellular iron accumulation, correlates strongly with senescence markers in MSC cultures under diverse conditions, including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues and can critically advance our understanding of cellular senescence.

Making shared decisions with older men selecting treatment for lower urinary tract symptoms secondary to benign prostatic hyperplasia (LUTS/BPH): a pilot randomized trial.

BACKGROUND: Making high-quality decisions when selecting treatment for lower urinary tract symptoms due to benign prostatic hyperplasia (LUTS/BPH) requires a shared decision-making approach. However, older people with lower health literacy face barriers. The pilot study aimed to evaluate the feasibility of recruiting participants and evaluate the effectiveness of a multi-level intervention on decision quality for the treatment of LUTS/BPH. METHOD: In this 2-arm, randomized controlled trial, multi-ethnic Asian men aged ≥ 50 years with moderate or severe symptoms (IPSS ≥ 8 and/or QOL ≥ 3) and physicians were recruited at a Singapore public primary care clinic. Men were randomized to either physicians trained in shared decision-making and used a pictorial patient-reported symptom score (Visual Analogue Uroflowmetry Score) during the consultation or to physicians untrained in shared decision-making who did not use the score. Decision quality was measured using SDMQ-9 scores from men and their physicians after the consultation. RESULTS: 60 men (intervention [n = 30], control [n = 30]) receiving care from 22 physicians were recruited. Men's mean age was 70 ± 9 years: 87% were Chinese, 40% had no formal education, and 32% were of lower socioeconomic status. No difference in decision quality from the men's nor their physicians' perspectives was noted [for men: mean score = 70.8 (SD 20.3) vs. 59.5 (SD 22.4); adjusted p = 0.352] [for physicians: mean score = 78.1 (SD 14.1) vs. 73.2 (SD 19.8); adjusted p > 0.999]. CONCLUSION: It was feasible to recruit the intended participants. There was no difference in decision quality between men who used shared decision-making and usual care for the treatment of LUTS/BPH.

Iatrogenic bladder injury from port insertion during laparoscopic appendicectomy.

A 26-year-old man underwent laparoscopic appendicectomy for acute appendicitis that was carried out uneventfully after initial urethral catheterisation to empty the bladder. Postoperatively, he developed oliguria associated with high drain output and elevated drain fluid creatinine. A contrast-enhanced computed tomography urography scan showed a small amount of contrast in the intraperitoneal space. A diagnostic laparoscopy performed for a suspected bladder injury revealed that the drain (inserted via the suprapubic port) had traversed the bladder. The drain was removed, and the bladder defects were repaired. The catheter was removed 2 weeks later uneventfully. It is important to recognise and avoid the urinary bladder during suprapubic port insertion during laparoscopic appendicectomy. This complication can be minimised via initial bladder decompression and introduction of the suprapubic port lateral to the umbilical ligaments. A high index of suspicion is required to diagnose a small bladder injury.

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells.

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.

Polymer-Based Precipitation of Extracellular Vesicular miRNAs from Serum Improve Gastric Cancer miRNA Biomarker Performance.

Circulating miRNAs are promising liquid biopsy biomarkers for noninvasive cancer detection. However, detection of subtle, but meaningful differences in circulating miRNA quantities between diseased and healthy samples remains a key challenge in clinical settings because biomarker signal/noise ratios are often low. Because extracellular vesicles (EVs) are key sources of circulating miRNAs in serum, it was hypothesized that isolating EVs would enrich miRNA biomarkers, leading to enhanced diagnostic ability and improved biomarker performance. This research assessed the performance of EV-miRNAs against serum miRNAs as biomarkers for gastric cancer (GC). It was first determined that polymer-based precipitation (PBP) gave the highest EV-miRNA recovery when compared with ultracentrifugation, column affinity, peptide affinity, and immunobead affinity EV purification. Four PBP reagents were used to isolate EV-miRNAs from 15 GC and 15 healthy controls and 133 GC-related miRNAs were profiled from EV fractions and total serum using real-time quantitative PCR. A PBP reagent that generated the most EV-miRNA biomarkers was selected and used to validate 11 EV-miRNAs in an independent set of 20 GC and 20 controls. Eight of these EV-miRNA biomarkers were found to give better GC detection accuracy (area under the curve, approximately 0.8). Overall, data suggest that EV miRNAs can improve GC detection performance compared with serum miRNAs and led to the identification of eight EV-miRNAs as potential noninvasive biomarkers for GC.

Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix.

The preservation of nucleic acids from clinical samples is critical to facilitate accurate molecular diagnosis. The use of a paper matrix, Flinders Technology Associates (FTA) Elute cards, to archive DNA and viral RNA is well-documented. However, the feasibility of FTA Elute cards for archiving serum and serum exosomal microRNAs (miRNAs) remains unclear. Here, we performed a comprehensive evaluation of FTA Elute cards for miRNA storage and recovery in different pre-analytical conditions. The recovery of serum miRNA dry-spotted on FTA Elute cards by direct elution with water at high temperature was poor. However, serum miRNAs dry-spotted on the cards were isolated with about 40% yield when using QIAzol lysis reagent and recovery was improved remarkably (>80%) upon extraction from cards pre-treated with trehalose. miRNAs stored on the cards remained stable at room temperature and can be kept for prolonged periods. Furthermore, miRNAs could be similarly recovered from serum exosomes dry-spotted on the cards. Importantly, when using sera from gastric cancer (GC) patients, the miRNAs were efficiently recovered from trehalose pre-treated cards without affecting their representation. Collectively, we have demonstrated the potential of FTA Elute cards to archive serum and serum exosomal miRNAs, making it useful for biomarker discovery and diagnostics.

Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells.

Use of a novel cytotoxic HEXIM1 peptide in the directed breast cancer therapy.

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb) and is recently identified as a novel positive regulator of p53. We previously showed the basic region (BR) of HEXIM1 mediates the binding of HEXIM1 to a nucleolar protein, nucleophosmin (NPM), and can be ubiquitinated by human double minute 2 protein. Here we identify a cytotoxic peptide derived from the BR of HEXIM1. When fused with a cell-penetrating peptide, the HEXIM1 BR peptide triggers rapid cytotoxic effect independent of p53. Similarly, when the BR peptide is linked with a breast cancer cell targeting peptide, LTV, the LTV-BR fusion peptide exhibits specific killing of breast cancer cells, which is not observed with the commonly used cytotoxic peptide, KLA. Importantly, the BR peptide fails to enter cells by itself and does not induce any cytotoxic effects when it is not guided by any cell-penetrating or cancer targeting peptides. We showed that HEXIM1 BR peptide depolarizes mitochondrial membrane potential in a p53-dependent manner and its cell-killing activity is not suppressed by caspase inhibition. Furthermore, we observed an accumulation of the internalized BR peptide in the nucleoli of treated cells and an altered localization of NPM. These results illustrate a novel mechanism which the BR peptide induces cell death and can potentially be used as a novel therapeutic strategy against breast cancer.

TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification.

The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification.

Assessment of sensory neuropathy in diabetic patients without diabetic foot problems.

OBJECTIVES: To study the incidence and extent of peripheral sensory neuropathy in diabetic patients without diabetic foot problems (DFPs) with <5, 5-10 and >10 years duration of diabetes using three different modalities of testing: Pin-Prick Testing, 5.07 Semmes-Weinstein Monofilament Testing (SWMT) and Rapid-Current Perception Threshold (R-CPT) measurements using the Neurometer. METHODS: Our study population consisted of 60 patients (120 feet) treated for diabetes mellitus in the Division of Endocrinology at the National University Hospital. No patient had any DFPs. Twenty-two, 21 and 17 patients had duration of diabetes of <5, 5-10 and >10 years, respectively. All patients were tested for sensory neuropathy using Pin-Prick Testing using a standardized protocol, SWMT and the Neurometer. RESULTS: There was a significantly higher incidence of sensory neuropathy detected by both the Pin-Prick Test and the Neurometer as compared to the SWMT. Also, in all three modalities, there was a significant increase in incidence of sensory neuropathy detected in diabetics with >5 years duration of diabetes. In addition, the Pin-Prick Test showed an increase in extent of sensory neuropathy with a longer duration of diabetes. CONCLUSIONS: The Pin-Prick Test was found to be a simple, cheap and useful diagnostic tool for detection of sensory neuropathy in diabetics without DFPs. In addition, it could accurately delineate the extent of neuropathy in the lower limb - additional useful information not obtainable with SWMT or Neurometer. Even for patients with <5 years duration of diabetes, the incidence of sensory neuropathy detected was considerable. The incidence of neuropathy detected continued to increase with length of duration of diabetes. Hence, we recommend screening of patients for neuropathy as soon as they are diagnosed with diabetes.