Procoagulant Activity of Umbilical Cord-Derived Mesenchymal Stromal Cells' Extracellular Vesicles (MSC-EVs).

Many cell types, including cancer cells, release tissue factor (TF)-exposing extracellular vesicles (EVs). It is unknown whether MSC-EVs pose a thromboembolism risk due to TF expression. Knowing that MSCs express TF and are procoagulant, we hypothesize that MSC-EVs also might. Here, we examined the expression of TF and the procoagulant activity of MSC-EVs and the impact of EV isolation methods and cell culture expansion on EV yield, characterization, and potential risk using a design of experiments methodology. MSC-EVs were found to express TF and have procoagulant activity. Thus, when MSC-derived EVs are employed as a therapeutic agent, one might consider TF, procoagulant activity, and thromboembolism risk and take steps to prevent them.

Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from Human Umbilical Cord-Derived Mesenchymal Stromal Cells.

EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, -20 °C, and -80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing -80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at -80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.

Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b+ macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death.

BACKGROUND: Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs. METHODS: C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs. RESULTS: Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix. CONCLUSIONS: These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

Mesenchymal Stem Cells Attenuate NADPH Oxidase-Dependent High Mobility Group Box 1 Production and Inhibit Abdominal Aortic Aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation, and matrix degradation. This study tests the hypothesis that macrophage-produced high mobility group box 1 (HMGB1) production is dependent on nicotinamide adenine dinucleotide phosphate oxidase (Nox2), which leads to increase in interleukin (IL)-17 production resulting in AAA formation and that treatment with human mesenchymal stem cells (MSCs) can attenuate this process thereby inhibiting AAA formation. APPROACH AND RESULTS: Human aortic tissue demonstrated a significant increase in HMGB1 expression in AAA patients when compared with controls. An elastase-perfusion model of AAA demonstrated a significant increase in HMGB1 production in C57BL/6 (wild-type [WT]) mice, which was attenuated by MSC treatment. Furthermore, anti-HMGB1 antibody treatment of WT mice attenuated AAA formation, IL-17 production, and immune cell infiltration when compared with elastase-perfused WT mice on day 14. Elastase-perfused Nox2(-/y) mice demonstrated a significant attenuation of HMGB1 and IL-17 production, cellular infiltration, matrix metalloproteinase activity, and AAA formation when compared with WT mice on day 14. In vitro studies showed that elastase-treated macrophages from WT mice, but not from Nox2(-/y) mice, produced HMGB1, which was attenuated by MSC treatment. The production of macrophage-dependent HMGB1 involved Nox2 activation and superoxide anion production, which was mitigated by MSC treatment. CONCLUSIONS: These results demonstrate that macrophage-produced HMGB1 leads to aortic inflammation and acts as a trigger for CD4(+) T-cell-produced IL-17 during AAA formation. HMGB1 release is dependent on Nox2 activation, which can be inhibited by MSCs leading to attenuation of proinflammatory cytokines, especially IL-17, and protection against AAA formation.

Inhibition of protein kinase C signaling maintains rat embryonic stem cell pluripotency.

Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.

Maternal Plasma RNA in First Trimester Nullipara for the Prediction of Spontaneous Preterm Birth ≤ 32 Weeks: Validation Study.

The first-trimester prediction of spontaneous preterm birth (sPTB) has been elusive, and current screening is heavily dependent on obstetric history. However, nullipara lack a relevant history and are at higher risk for spontaneous (s)PTB ≤ 32 weeks compared to multipara. No available objective first-trimester screening test has proven a fair predictor of sPTB ≤ 32 weeks. We questioned whether a panel of maternal plasma cell-free (PCF) RNAs (PSME2, NAMPT, APOA1, APOA4, and Hsa-Let-7g) previously validated at 16-20 weeks for the prediction of sPTB ≤ 32 weeks might be useful in first-trimester nullipara. Sixty (60) nulliparous women (40 with sPTB ≤ 32 weeks) who were free of comorbidities were randomly selected from the King's College Fetal Medicine Research Institute biobank. Total PCF RNA was extracted and the expression of panel RNAs was quantitated by qRT-PCR. The analysis employed, primarily, multiple regression with the main outcome being the prediction of subsequent sPTB ≤ 32 weeks. The test performance was judged by the area under the curve (AUC) using a single threshold cut point with observed detection rates (DRs) at three fixed false positive rates (FPR). The mean gestation was 12.9 ± 0.5 weeks (range 12.0-14.1 weeks). Two RNAs were differentially expressed in women destined for sPTB ≤ 32 weeks: APOA1 (p < 0.001) and PSME2 (p = 0.05). APOA1 testing at 11-14 weeks predicted sPTB ≤ 32 weeks with fair to good accuracy. The best predictive model generated an AUC of 0.79 (95% CI 0.66-0.91) with observed DRs of 41%, 61%, and 79% for FPRs of 10%, 20%, and 30%, including crown-rump length, maternal weight, race, tobacco use, and age.

Detection of Embryonic Trisomy 21 in the First Trimester Using Maternal Plasma Cell-Free RNA.

Prenatal trisomy 21 (T21) screening commonly involves testing a maternal blood sample for fetal DNA aneuploidy. It is reliable but poses a cost barrier to universal screening. We hypothesized maternal plasma RNA screening might provide similar reliability but at a lower cost. Discovery experiments used plasma cell-free RNA from 20 women 11−13 weeks tested by RNA and miRNA microarrays followed by qRT-PCR. Thirty-six mRNAs and 18 small RNAs of the discovery cDNA were identified by qPCR as potential markers of embryonic T21. The second objective was validation of the RNA predictors in 998 independent pregnancies at 11−13 weeks including 50 T21. Initial analyses identified 9−15 differentially expressed RNA with modest predictive power (AUC < 0.70). The 54 RNAs were then subjected to machine learning. Eleven algorithms were trained on one partition and tested on an independent partition. The three best algorithms were identified by Kappa score and the effects of training/testing partition size and dataset class imbalance on prediction were evaluated. Six to ten RNAs predicted T21 with AUCs up to 1.00. The findings suggest that maternal plasma collected at 11−13 weeks, tested by qRT-PCR, and classified by machine learning, may accurately predict T21 for a lower cost than plasma DNA, thus opening the door to universal screening.

Therapeutic Use of Mesenchymal Stromal Cells: The Need for Inclusive Characterization Guidelines to Accommodate All Tissue Sources and Species.

Following their discovery over 50 years ago, mesenchymal stromal cells (MSCs) have become one of the most studied cellular therapeutic products by both academia and industry due to their regenerative potential and immunomodulatory properties. The promise of MSCs as a therapeutic modality has been demonstrated by preclinical data yet has not translated to consistent, successful clinical trial results in humans. Despite the disparities across the field, MSC shareholders are unified under one common goal-to use MSCs as a therapeutic modality to improve the quality of life for those suffering from a malady in which the standard of care is suboptimal or no longer effective. Currently, there is no Food and Drug Administration (FDA)-approved MSC therapy on the market in the United States although several MSC products have been granted regulatory approval in other countries. In this review, we intend to identify hurdles that are impeding therapeutic progress and discuss strategies that may aid in accomplishing this universal goal of widespread therapeutic use.

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis.

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors). Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)'s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

A Protocol for the Isolation, Culture, and Cryopreservation of Umbilical Cord-Derived Canine Mesenchymal Stromal Cells: Role of Cell Attachment in Long-Term Maintenance.

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn's disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. By review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared with human and rodent MSCs, and limited lifespan is an obstacle for preclinical investigation and therapeutic use. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories are problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average >90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.

A Phase I Study to Evaluate Two Doses of Wharton's Jelly-Derived Mesenchymal Stromal Cells for the Treatment of De Novo High-Risk or Steroid-Refractory Acute Graft Versus Host Disease.

BACKGROUND: Because of their well-described immunosuppressive properties, allogeneic adult human mesenchymal stromal cells (MSC) derived from bone marrow have demonstrated safety and efficacy in steroid refractory acute graft versus host disease (SR aGVHD). Clinical trials have resulted in variable success and an optimal source of MSC has yet to be defined. Based on the importance of maternal-fetal interface immune tolerance, extraembryonic fetal tissues, such as the umbilical cord, may provide an superior tissue source of MSC to mediate immunomodulation in aGVHD. METHODS: A two-dose cohort trial allogeneic Wharton's Jelly-derived mesenchymal stromal cells (WJMSC, referred to as MSCTC-0010, here) were tested in 10 patients with de novo high risk (HR) or SR aGVHD post allogeneic hematopoietic stem cell transplantation (allo-HCT). Following Good Manufacturing Practices isolation, expansion and cryostorage, WJMSC were thawed and administered via intravenous infusions on days 0 and 7 at one of two doses (low dose cohort, 2 × 106/kg, n = 5; high dose cohort, 10 × 106/kg, n = 5). To evaluate safety, patients were monitored for infusion related toxicity, Treatment Related Adverse Events (TRAE) til day 42, or ectopic tissue formation at day 90. Clinical responses were monitored at time points up to 180 days post infusion. Serum biomarkers ST2 and REG3α were acquired 1 day prior to first MSCTC-0010 infusion and on day 14. RESULTS: Safety was indicated, e.g., no infusion-related toxicity, no development of TRAE, nor ectopic tissue formation in either low or high dose cohort was observed. Clinical response was suggested at day 28: the overall response rate (ORR) was 70%, 4 of 10 patients had a complete response (CR) and 3 had a partial response (PR). By study day 90, the addition of escalated immunosuppressive therapy was necessary in 2 of 9 surviving patients. Day 100 and 180 post infusion survival was 90% and 60%, respectively. Serum biomarker REG3α decreased, particularly in the high dose cohort, and with REG3α decrease correlated with clinical response. CONCLUSIONS: Treatment of patients with de novo HR or SR aGVHD with low or high dose MSCTC-0010 was safe: the infusion was well-tolerated, and no TRAEs or ectopic tissue formation was observed. A clinical improvement was seen in about 70% patients, with 4 of 10 showing a complete response that may have been attributable to MSCTC-0010 infusions. These observations indicate safety of two different doses of MSCTC-0010, and suggest that the 10 × 106 cells/ kg dose be tested in an expanded randomized, controlled Phase 2 trial. Graphical abstract.

Wharton's jelly-derived cells are a primitive stromal cell population.

Here, the literature was reviewed to evaluate whether a population of mesenchymal stromal cells derived from Wharton's jelly cells (WJCs) is a primitive stromal population. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. In contrast to bone marrow MSCs, WJCs have greater expansion capability, faster growth in vitro, and may synthesize different cytokines. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. The preclinical work suggests that the WJCs are therapeutic via trophic rescue and immune modulation. In summary, WJCs meet the definition of MSCs. Since WJCs expand faster and to a greater extent than adult-derived MSCs, these findings suggest that WJCs are a primitive stromal cell population with therapeutic potential. Further work is needed to determine whether WJCs engraft long-term and display self-renewal and multipotency in vivo and, as such, demonstrate whether Wharton's jelly cells are a true stem cell population.

Immune properties of human umbilical cord Wharton's jelly-derived cells.

Cells isolated from Wharton's jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue-culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self-renew, and are multipotent (differentiate into bone, fat, cartilage, etc.) in vitro. These properties support the notion that UCMS cells are a member of the MSC family. Here, the immune properties of UCMS cells are characterized in vitro. The overall hypothesis is that UCMS cells possess immune properties that would be permissive to allogeneic transplantation. For example, UCMS cells will suppress of the proliferation of "stimulated" lymphocytes (immune suppression) and have reduced immunogenicity (e.g., would be poor stimulators of allogeneic lymphocyte proliferation). Hypothesis testing was as follows: first, the effect on proliferation of coculture of mitotically inactivated human UCMS cells with concanavalin-A-stimulated rat splenocytes was assessed in three different assays. Second, the effect of human UCMS cells on one-way and two-way mixed lymphocyte reaction (MLR) assays was determined. Third, the expression of human leukocyte antigen (HLA)-G was examined in human UCMS cells using reverse transcription-polymerase chain reaction, since HLA-G expression conveys immune regulatory properties at the maternal-fetal interface. Fourth, the expression of CD40, CD80, and CD86 was determined by flow cytometry. Fifth, the cytokine expression of UCMS cells was evaluated by focused gene array. The results indicate that human UCMS cells inhibit splenocyte proliferation response to concanavalin A stimulation, that they do not stimulate T-cell proliferation in a one-way MLR, and that they inhibit the proliferation of stimulated T cells in a two-way MLR. Human UCMS cells do not inhibit nonstimulated splenocyte proliferation, suggesting specificity of the response. UCMS cells express mRNA for pan-HLA-G. UCMS cells do not express the costimulatory surface antigens CD40, CD80, and CD86. UCMS cells express vascular endothelial growth factor and interleukin-6, molecules previously implicated in the immune modulation observed in MSCs. In addition, the array data indicate that UCMS cells make a cytokine and other factors that may support hematopoiesis. Together, these results support previous observations made following xenotransplantation; for example, there was no evidence of frank immune rejection of undifferentiated UCMS cells. The results suggest that human UCMS will be tolerated in allogeneic transplantation. Disclosure of potential conflicts of interest is found at the end of this article.

Defining hydrogel properties to instruct lineage- and cell-specific mesenchymal differentiation.

The maintenance and direction of stem cell lineage after implantation remains challenging for clinical translation. Aggregation and encapsulation into instructive biomaterials after preconditioning can bolster retention of differentiated phenotypes. Since these procedures do not depend on cell type or lineage, we hypothesized we could use a common, tunable platform to engineer formulations that retain and enhance multiple lineages from different cell populations. To test this, we varied alginate stiffness and adhesive ligand content, then encapsulated spheroids of varying cellularity. We used Design-of-Experiments to determine the effect of these parameters and their interactions on phenotype retention. The combination of parameters leading to maximal differentiation varied with lineage and cell type, inducing a 2-4-fold increase over non-optimized levels. Phenotype was also retained for 4 weeks in a murine subcutaneous model. This widely applicable approach can facilitate translation of cell-based therapies by instructing phenotype in situ without prolonged induction or costly growth factors.

Combination treatment of human umbilical cord matrix stem cell-based interferon-beta gene therapy and 5-fluorouracil significantly reduces growth of metastatic human breast cancer in SCID mouse lungs.

Umbilical cord matrix stem (UCMS) cells that were engineered to express interferon-beta (IFN-beta) were transplanted weekly for three weeks into MDA 231 breast cancer xenografts bearing SCID mice in combination with 5-fluorouracil (5-FU). The UCMS cells were found within lung tumors but not in other tissues. Although both treatments significantly reduced MDA 231 tumor area in the SCID mouse lungs, the combined treatment resulted in a greater reduction in tumor area than by either treatment used alone. These results indicate that a combination treatment of UCMS-IFN-beta cells and 5-FU is a potentially effective therapeutic procedure for breast cancer.

Human Umbilical Cord Mesenchymal Stromal Cell Isolation, Expansion, Cryopreservation, and Characterization.

Revised methods to derive, expand, and characterize mesenchymal stromal cells (MSCs) from the umbilical cord are provided. Several considerations are taken for GMP compliance including using a closed system isolation method and eliminating several xenogenic components. With this method cells are isolated using mechanical and enzymatic digestion and then expanded with high viabilities that retain >90% viability after cryopreservation. Lastly, characterization methods have been optimized to identify these cells as MSCs according to the ISCT minimal criteria. This method standardizes the process for isolating, expanding, cryopreserving, and characterizing MSCs from the umbilical cord. © 2017 by John Wiley & Sons, Inc.

Effects of cyclosporine-A on rat soleus muscle fiber size and phenotype.

PURPOSE: Organ transplant patients treated with cyclosporine-A (CsA) often exhibit weight loss and muscle weakness. The cellular target of CsA, calcineurin, has been implicated in maintenance of muscle fiber size and in expression of the type I skeletal muscle phenotype. We hypothesized that CsA treatment would cause fiber atrophy, as well as increase type IIa myosin heavy chain (MHC) content and oxidative enzyme activities in the soleus muscle. METHODS: Rats were treated with CsA for 21 d (20 mg.kg(-1).d(-1); N = 16) and compared with control rats given olive oil vehicle (Veh; N = 16). Soleus muscles were excised bilaterally. MHC content was determined by gel electrophoresis, oxidative enzyme activities by spectrophotometric methods, and fiber type and size by histochemistry. RESULTS: Lymphocyte count was depressed in CsA rats (P < 0.05), indicating treatment efficacy. Type IIa MHC content was increased in the soleus muscle with CsA (Veh, 10.4 +/- 1.7%; CsA, 15.1 +/- 2.0; P < 0.05) at the expense of type I MHC. Soleus muscle oxidative enzyme activities were also increased with CsA treatment (P < 0.05). Soleus muscle atrophy occurred, reflected by a 22% decrease in fiber cross-sectional area (Veh, 3255 +/- 105 microm(2); CsA, 2533 +/- 125; P < 0.05). CONCLUSION: These findings indicate that CsA treatment is associated with changes in skeletal muscle fiber size and phenotype. The former may underlie clinical symptoms of transplant patients treated with CsA.

Stem cells in the umbilical cord.

Stem cells are the next frontier in medicine. Stem cells are thought to have great therapeutic and biotechnological potential. This will not only to replace damaged or dysfunctional cells, but also rescue them and/or deliver therapeutic proteins after they have been engineered to do so. Currently, ethical and scientific issues surround both embryonic and fetal stem cells and hinder their widespread implementation. In contrast, stem cells recovered postnatally from the umbilical cord, including the umbilical cord blood cells, amnion/placenta, umbilical cord vein, or umbilical cord matrix cells, are a readily available and inexpensive source of cells that are capable of forming many different cell types (i.e., they are "multipotent"). This review will focus on the umbilical cord-derived stem cells and compare those cells with adult bone marrow-derived mesenchymal stem cells.

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.

Umbilical cord derived mesenchymal stromal cells (UC-MSCs) are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP) for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed-vessel dissociation following enzymatic digestion which reduces contamination risk and manipulation time. The new method produced >10 times more cells per cm of UC than our previous method. When biographical variables were compared, more UC-MSCs per gram were isolated after vaginal birth compared to Caesarian-section births, an unexpected result. UC-MSCs were expanded in medium enriched with 2%, 5%, or 10% pooled human platelet lysate (HPL) eliminating the xenogeneic serum components. When the HPL concentrations were compared, media supplemented with 10% HPL had the highest growth rate, smallest cells, and the most viable cells at passage. UC-MSCs grown in 10% HPL had surface marker expression typical of MSCs, high colony forming efficiency, and could undergo trilineage differentiation. The new protocol standardizes manufacturing of UC-MSCs and enables clinical translation.