Stem Cell Derived Extracellular Vesicle Therapy for Multiple Sclerosis, A Systematic Review and Meta-Analysis of Preclinical Studies.

Stem cell therapy holds promise for multiple sclerosis (MS), with efficacy of different stem cell types reported across a range of preclinical MS animal models. While stem cell therapy has been approved for a small number of diseases in humans, extracellular vesicles (EVs) may provide an efficacious, cost-effective, and safer alternative to stem cell therapy. To this end, we conducted a systematic review with meta-analysis to assess the effectiveness of stem cell-derived secretome (EV and conditioned media (CM)) in animal models of MS. The data were extracted to calculate standardized mean differences for primary outcome measure of disease severity, using a random effect model. Additionally, several subgroup analyses were conducted to assess the impact of various study variables such as stem cell type and source, stem cell modification, route and time of administration, number of animals and animal's age, and EV isolation methods on secondary outcome. Publication quality and risk of bias were assessed. Overall, 19 preclinical studies were included in the meta-analysis where stem cell EV/CM was found to significantly reduce disease severity in EV-treated (SMD = 2, 95% CI: 1.18-2.83, P < .00001) and CM-treated animals (SMD = 2.58, 95% CI: 1.34-3.83, P < .00001) compared with controls. Our analysis indicated that stem cell secretome has a positive effect on reducing demyelination, systemic neuroinflammation, and disease severity in preclinical models of MS. These findings indicate a potential therapeutic effect that merits investigation and validation in clinical settings.

Implications of maternal-fetal health on perinatal stem cell banking.

Cell based therapies are being assessed for their therapeutic potential across a variety of diseases. Gestational tissues are attractive sources for cell therapy. The large number of births worldwide ensures sufficient access to gestational tissues, however, limited information has been reported around the impact of birth trends, delivery methods and pregnancy conditions on perinatal stem cell banking. This review describes the current state of banking of gestational tissues and their derived perinatal stem cells, discusses why the changes in birth trends and delivery methods could affect gestational tissue banking practices, and further explores how common pregnancy complications can potentially influence perinatal stem cell banking.

Assessing the impact of gestational age of donors on the efficacy of amniotic epithelial cell-derived extracellular vesicles in experimental bronchopulmonary dysplasia.

BACKGROUND AND RATIONALE: Extracellular vesicles (EVs) are a potential cell-free regenerative medicine. Human amniotic epithelial cells (hAECs) are a viable source of cell therapy for diseases like bronchopulmonary dysplasia (BPD). However, little is known about the impact of gestational age of the donor on the quality of hAEC-derived EVs. AIMS: To determine the impact of gestational age on hAEC-derived EVs in experimental BPD. RESULTS: Term hAEC-derived EVs displayed a significantly higher density of surface epitopes (CD142 and CD133) and induced greater macrophage phagocytosis compared to preterm hAEC-EVs. However, T cell proliferation was more significantly suppressed by preterm hAEC-EVs. Using a model of experimental BPD, we observed that term but not preterm hAEC-EVs improved tissue-to-airspace ratio and septal crest density. While both term and preterm hAEC-EVs reduced the levels of inflammatory cytokines on postnatal day 7, the improvement in lung injury was associated with increased type II alveolar cells which was only observed in term hAEC-EV treatment group. Furthermore, only neonatal term hAEC-EVs reduced airway hyper-responsiveness, mitigated pulmonary hypertension and protected against right ventricular hypertrophy at 6 weeks of age. CONCLUSION: Term hAEC-EVs, but not preterm hAEC-EVs, have therapeutic efficacy in a mouse model of BPD-like lung injury. Therefore, the impact of donor criteria should be considered when applying perinatal cells-derived EV therapy for clinical use.

Improving cell viability using counterflow centrifugal elutriation.

BACKGROUND: Cell viability is an important release criterion in the manufacturing of cell therapy products. Low cell viability can have significant impact on product quality and manufacturing efficiency. Counterflow centrifugation technology has been applied to the manufacturing of cell therapy products, to enable cell separation based on size and density. This study evaluated the utility of counterflow centrifugation technology for dead cell removal to improve cell viability of the final product. METHODS: Jurkat cell cultures with low and high dead cell burden were subjected to counterflow centrifugal elutriation to determine the correlation between process parameters (e.g., flow rate, centrifugal force) and processing outcomes (i.e., cell recovery and viability). Subsequently, the optimized parameters were applied to dead cell elutriation using expanded T cells and freshly isolated human amniotic epithelial cells (hAECs). The efficiency of dead cell removal, cell function and post-thaw viability were compared. RESULTS: Elutriation using a low flow rate allowed better control of viable cell recovery from both low and high dead cell burden cultures of Jurkat cells. The viability of T cells and hAECs was improved by counterflow centrifugal processing, from 80.67% ± 2.33 to 94.73% ± 1.19 and 79.19% ± 5.35 to 90.34% ± 3.59, respectively. Processing increased the proliferation rate of T cells, while the metabolic activity of hAECs was unchanged. CONCLUSION: Counterflow centrifugal elutriation can be added as an integrated step to the automated wash-and-concentrate protocol for cell manufacturing to remove dead cells and improve cell viability of the final product.

Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells.

The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs.

Automated Counterflow Centrifugal System for Small-Scale Cell Processing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Dynamic variation of kidney injury molecule-1 mRNA and protein expression in blood and urine of renal transplant recipients: a cohort study.

BACKGROUND: Acute renal dysfunction still constitutes a highly significant obstacle to renal transplantation outcome. Kidney injury molecule-1 is highly upregulated in proximal tubular cells and shed into the urine and blood circulation following kidney injury. The aim of current cohort study was to evaluate the urine KIM-1 (uKIM-1) mRNA expression level and its protein concentration in blood and urine samples to determine whether sequential monitoring of KIM-1 in renal allograft recipients is a reliable biomarker for predicting the clinical status and outcome. METHODS: Both uKIM-1 mRNA expression level and the level of serum and uKIM-1 protein concentration in the 52 renal transplant recipients were respectively quantified using real-time PCR and ELISA methods at 2, 90 and 180 days after transplantation. RESULT: KIM-1 mRNA and protein expression level in the blood and urine samples of patients with graft dysfunction was significantly higher than patients with well-functioning graft on days 2, 90 and 180 after transplantation. Receiver-operating characteristic curve analysis of mRNA and protein expression levels showed that urinary and blood KIM-1 at months 3 and 6 could predict acute renal dysfunction at 6 months and 1 year after transplantation. CONCLUSION: Sequential monitoring of uKIM-1 mRNA expression level and its protein concentration in the serum and urine samples of renal transplant patients suggests that KIM-1 could be a sensitive and specific biomarker for early diagnosis and prognosis of kidney allograft injury.

Sequential monitoring of TIM-3 mRNA expression in blood and urine samples of renal transplant recipients.

BACKGROUND: T cell immunoglobulin and mucin domain 3 (TIM-3), as a co-inhibitory receptor expressed on Th1, Th17, CD8T, FoxP3 + Treg and innate immune cells, plays an important role in suppression of T cell-mediated immune responses, tolerance induction and T cell exhaustion. In this study, we evaluated sequential alterations of TIM-3 mRNA expression level in blood and urine samples of renal transplant recipients to predict approaching clinical episodes. METHODS: A total of 52 adult renal transplant recipients (31 male and 21 female) were enrolled in this study. All the patients received kidney transplant from living unrelated donors. TIM-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) and urinary cells were quantified using Real Time TaqMan polymerase chain reaction (PCR) at 4 different time points (pre-transplantation, 2, 90 and 180 days post-transplantation). RESULT: TIM-3 mRNA expression level on days 2, 90 and 180 after transplantation was significantly higher in blood and urine samples of patients with graft dysfunction (GD) compared with patients with well-functioning graft (WFG). Our results also showed a high correlation between blood and urinary level of TIM-3 mRNA expression. The data from Receiver Operating Characteristic (ROC) Curve Analysis showed that blood and urinary TIM-3 mRNA expression level at month 3 and 6 could discriminate graft dysfunction (GD) from well-functioning graft (WFG) with high specificity and sensitivity. CONCLUSION: Our data suggested that serial monitoring of TIM-3 mRNA level in the blood and urine samples of renal transplant recipients could be a useful non-invasive biomarker for prediction and diagnosis of allograft dysfunction.

To Protect and to Preserve: Novel Preservation Strategies for Extracellular Vesicles.

Extracellular vesicles (EVs)-based therapeutics are based on the premise that EVs shed by stem cells exert similar therapeutic effects and these have been proposed as an alternative to cell therapies. EV-mediated delivery is an effective and efficient system of cell-to-cell communication which can confer therapeutic benefits to their target cells. EVs have been shown to promote tissue repair and regeneration in various animal models such as, wound healing, cardiac ischemia, diabetes, lung fibrosis, kidney injury, and many others. Given the unique attributes of EVs, considerable thought must be given to the preservation, formulation and cold chain strategies in order to effectively translate exciting preclinical observations to clinical and commercial success. This review summarizes current understanding around EV preservation, challenges in maintaining EV quality, and also bioengineering advances aimed at enhancing the long-term stability of EVs.

Potential role of RAB6C-AS1 long noncoding RNA in different cancers.

BACKGROUND: Long noncoding RNAs (lncRNAs) refer to a group of non-protein-coding RNAs that are usually more than 200 nucleotides. These long transcripts play significant roles in diverse cellular processes, mostly through epigenetic mechanisms. Thus, dysregulation of lncRNAs is associated with various diseases, especially cancer. This study aims to investigate the probable roles of RAB6C-AS1 lncRNA in different cancers. METHODS: Real-time quantitative reverse transcription-polymerase chain reaction was applied for the analysis of RAB6C-AS1 lncRNA amplification in gastric cancer (GC) samples compared with normal ones. Also, several online and offline data sets and tools were used to analyze the relation between RAB6C-AS1 lncRNA and different cancers. RESULTS: The end result of our analyses indicated that RAB6C-AS1 was overexpressed in 40% of the investigated GC specimens. Also, the results demonstrated a true relation between RAB6C-AS1 overexpression and higher GC tumor grades. However, bioinformatic analyses showed that while RAB6C-AS1 possibly functions as an oncogene in some cancer types, including prostate and breast cancers, it might have a tumor suppressive function in some others including brain tumors. CONCLUSIONS: We found that RAB6C-AS1 lncRNA is mostly overexpressed in GC. Also, based on bioinformatic and systems biology analyses, RAB6C-AS1 might function either as an oncogenic factor or tumor suppressor in a tissue-specific manner. Thus, RAB6C-AS1 could be considered as a candidate biomarker for various malignancies, especially prostate and brain cancers. According to our results, RAB6C-AS1 has a notable prognostic value for patients with brain lower grade glioma.

High Expression of FOXP3 mRNA in Blood and Urine as a Predictive Marker in Kidney Transplantation.

BACKGROUND: Diagnosis of allograft dysfunction by noninvasive biomarker tests is preferable to invasive allograft biopsies and has been extensively considered in recent years. This study aims to evaluate blood and urinary forkhead box P3 (FOXP3) messenger RNA (mRNA) expression in renal transplant recipients in an attempt to determine whether differential diagnosis of graft dysfunction is feasible using mRNA profiles. METHODS: We analyzed FOXP3 mRNA expression in paired urinary and peripheral blood mononuclear cell (PBMC) samples. A total of 91 kidney transplant recipients enrolled in this study that were classified into 3 groups: biopsy-proven acute rejection (AR; n = 27), chronic allograft nephropathy (n = 19), and well-functioning graft (n = 45). The FOXP3 mRNA expression was quantified by TaqMan probe real-time polymerase chain reaction. RESULTS: Acute rejection patients had a higher expression level of transcription factor FOXP3 compared to the chronic nephropathy and control groups. Analysis of receiver operating characteristic curves showed that rejection could be diagnosed with 100% sensitivity and 96% specificity in urine, and 92% sensitivity and 86% specificity in PBMC samples using the optimal FOXP3 mRNA cutoff value. We subdivided the AR group into progressive and nonprogressive patients, which showed a significant difference in FOXP3 mRNA expression. This result confirmed the role of FOXP3 as a diagnostic marker in predicting transplantation outcomes. CONCLUSION: Our results suggested that elevated expression of FOXP3 in blood and urine samples from kidney transplant recipients could be a useful noninvasive biomarker to diagnose graft dysfunction.

Expression patterns of Toll like receptor (TLR)-2, TLR-4 and myeloid differentiation primary response gene 88 (MYD88) in renal transplant patients developing allograft dysfunction; a cohort study.

This cohort intends to determine the sequential dynamic changes in Toll-like receptor (TLR)-4, TLR-2, and myeloid differentiation primary response gene 88 (MYD88) mRNA expressions in PBMCs and biopsy samples from kidney allograft recipients in relation to graft function. This study enrolled 52 renal transplant patients, 27 with well functioning graft (WFG) and 25 graft dysfunction (GD). Peripheral blood samples pre- and post-transplantation (days 2, 90 and 180) were collected to analyze mRNA expression levels of TLR-2, TLR-4, and MYD88 genes in relation to allograft function during one-year follow up. The mean dynamic changes of post-transplant TLR-2, TLR-4, and MYD88 mRNA expressions were significantly higher in GD compared to WFG patients (P = .001). ROC curve analysis based on glomerular filtration rate (GFR) index showed the area under curve (AUC) values for the genes: TLR-2(0.89;P < .001), TLR-4(0.86;P < .001), and MYD88(0.75;P = .003) in the third month post-transplantation for GD diagnosis. The calculated AUCs for the expressions of genes in allograft biopsies were 0.94(TLR-2), 0.95(TLR-4), and 0.98(MYD88) in the sixth month post-transplant based on pathology report (P < .001). Our results indicate that sequential monitoring of the expression patterns of TLR-2, TLR-4, and MYD88 in PBMCs and biopsy samples could be considered as predictive biomarkers for early and late kidney allograft function.

High expression of TIM-3 and KIM-1 in blood and urine of renal allograft rejection patients.

BACKGROUND: T cell immunoglobulin and mucin domain 3 (TIM-3) is involved in alloimmune and autoimmune responses, as well as tolerance induction in kidney transplantation. Kidney injury molecule-1 (KIM-1) is highly expressed in epithelial cells of the injured proximal tubule. In this study, we have investigated both urinary and blood TIM-3 mRNA expressions, urinary KIM-1 mRNA expression, and urinary and serum KIM-1 proteins in renal allograft recipients diagnosed with acute allograft rejection (AR) and chronic allograft dysfunction (CAD), as well as those with well-functioning transplants (WFG). METHODS: We divided 85 patients into the following groups: AR (n=24), CAD (n=19), and WFG (n=42). TIM-3 and KIM-1 mRNA expressions were quantified using real-time reverse-transcription TaqMan probe polymerase chain reaction (RT-PCR). An ELISA test was used to measure the amount of KIM-1 protein in serum and urine samples. RESULTS: AR and CAD patients had significantly greater urinary and blood TIM-3 mRNA expressions, urinary KIM-1 mRNA expression, and urinary and serum KIM-1 proteins compared to WFG patients. Receiver operating characteristic (ROC) analysis showed that these molecules discriminated Allograft rejections from WFG. CONCLUSION: Quantification of TIM-3 and KIM-1 mRNA expressions, along with KIM-1 protein measurements in urine and blood could be employed as promising tools for noninvasive diagnosis of allograft dysfunction.

Increased levels of CD4(+) and CD8(+) T cells expressing CCR1 in patients developing allograft dysfunction; a cohort study.

BACKGROUND: Leukocyte infiltration into the graft has pivotal effects on kidney transplantation outcome. The present study sought to determine whether the expression of sequential chemokine receptors on CD4(+) and CD8(+) T cells in human renal allograft can predict clinical episodes. METHODS: Blood samples from 52 consecutive renal transplant patients were evaluated at the time of transplantation and at three times (2, 90 and 180days) after transplantation to analyze the expression of CCR1 and CXCR3 on CD4(+) and CD8(+) T cells by flowcytometry. A total of 30 biopsies, including protocol biopsy (n=24) and cause biopsy (n=6), were investigated according to the Banff criteria. RESULTS: The mean percentage of CD4(+) and CD8(+) T cells expressing CCR1 was significantly increased in patients with allograft dysfunction (n=25) (p=0.006, p=0.004). The mean fluorescence intensity of CXCR3 on CD4(+) and CD8(+) T cells were found to be significantly higher in graft dysfunction than that in well-functioning grafts. (p<0.001, p=0.007). Receiver Operating Characteristic (ROC) Curve Analysis showed that the calculated AUC was 0.86 at the third month for CD4(+)CCR1(+) and CD8(+)CCR1(+) (p<0.001). Multiple logistic regression analysis showed that an increase in CD4(+) expressing CXCR3 leads to a lower risk of graft dysfunction (OR=0.37), while an increase in CD8(+) expressing CCR1 results in a higher risk of graft dysfunction (OR=3.66). CONCLUSION: During renal transplantation, CD4(+) and CD8(+) T cells expressing CCR1 were increased in patients who developed graft dysfunction. These findings may prospectively predict allograft dysfunction, and help elucidate the underlying pathogenic mechanisms.

Altered Expression of MicroRNAs Following Chronic Allograft Dysfunction with Interstitial Fibrosis and Tubular Atrophy.

Chronic allograft dysfunction (CAD) remains the major cause of renal transplant loss and characterized by interstitial fibrosis and tubular atrophy (IFTA). MicroRNAs (miRNAs) are implicated in many biological processes as well as innate and adaptive immune responses. We aimed to investigate whether CAD with IFTA is associated with differential expression of miR-142-5p, miR-142-3p and miR-211 within biopsy and peripheral blood mononuclear cell (PBMC) samples and whether expression of miRNAs are diagnostic for CAD with IFTA and predicts renal allograft function. In this study, biopsy and PBMC samples of 16 CAD with IFTA and 17 normal allografts (NA) were collected. Using Taqman MicroRNA Assays the expression levels of miR-142-5p, miR-142-3p and miR-211 were determined in two groups. Our results showed that miR-142-5p and miR-142-3p were significantly (p<0.0001) up-regulated and miR-211 was significantly (p<0.0001) down-regulated in renal allograft tissues of CAD with IFTA compared with NA recipients. Moreover, miR-142-3p and miR-211 were significantly (p<0.0001) up-regulated and down-regulated respectively in PBMC samples of CAD with IFTA. According to the ROC curve analysis, miR-142-5p in biopsy samples, but miR-142-3p and miR-211 both in biopsy and PBMC samples could be used as a diagnostic biomarker of CAD with IFTA and a prediction factor of allograft function. In this study, miRNAs were differentially expressed in the kidney allograft biopsy and simultaneously in PBMC samples of patients with CAD with IFTA. We suggest that the expression of miRNAs in PBMC might be used for monitoring the post transplantation and also as potential non-invasive biomarkers of kidney graft function and CAD with IFTA.