Uncovering Novel Protein Partners of Inducible Nitric Oxide Synthase in Human Testis.

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.

Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction.

Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE2) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.

Human-derived Treg and MSC combination therapy may augment immunosuppressive potency in vitro, but did not improve blood brain barrier integrity in an experimental rat traumatic brain injury model.

Traumatic brain injury (TBI) causes both physical disruption of the blood brain barrier (BBB) and altered immune responses that can lead to significant secondary brain injury and chronic inflammation within the central nervous system (CNS). Cell therapies, including mesenchymal stromal cells (MSC), have been shown to restore BBB integrity and augment endogenous splenic regulatory T cells (Treg), a subset of CD4+ T cells that function to regulate immune responses and prevent autoimmunity. We have recently shown that infusion of human cord blood-derived Treg decreased neuroinflammation after TBI in vivo and in vitro. However, while both cells have demonstrated anti-inflammatory and regenerative potential, they likely utilize differing, although potentially overlapping, mechanisms. Furthermore, studies investigating these two cell types together, as a combination therapy, are lacking. In this study, we compared the ability of Treg+MSC combination therapy, as well as MSC and Treg monotherapies, to improve BBB permeability in vivo and suppress inflammation in vitro. While Treg+MSC combination did not significantly augment potency in vivo, our in vitro data demonstrates that combination therapy may augment therapeutic potency and immunosuppressive potential compared to Treg or MSC monotherapy.

Combination therapy with Treg and mesenchymal stromal cells enhances potency and attenuation of inflammation after traumatic brain injury compared to monotherapy.

The inflammatory response after traumatic brain injury (TBI) can lead to significant secondary brain injury and chronic inflammation within the central nervous system. Cell therapies, including mesenchymal stromal cells (MSC), have led to improvements in animal models of TBI and are under investigation in human trials. One potential mechanism for the therapeutic potential of MSC is their ability to augment the endogenous response of immune suppressive regulatory T cells (Treg). We have recently shown that infusion of human cord blood Treg decreased chronic microgliosis after TBI and altered the systemic immune response in a rodent model. These cells likely use both overlapping and distinct mechanisms to modulate the immune system; therefore, combining Treg and MSC as a combination therapy may confer therapeutic benefit over either monotherapy. However, investigation of Treg + MSC combination therapy in TBI is lacking. In this study, we compared the ability MSC + Treg combination therapy, as well as MSC and Treg monotherapies, to inhibit the neuroinflammatory response to TBI in vivo and in vitro. Treg + MSC combination therapy demonstrated increased potency to reduce the neuro- and peripheral inflammatory response compared to monotherapy; furthermore, the timing of infusion proved to be a significant variable in the efficacy of both MSC monotherapy and Treg + MSC combination therapy in vivo and in vitro.

Human adipose-derived mesenchymal stem cells for acute and sub-acute TBI.

In the U.S., approximately 1.7 million people suffer traumatic brain injury each year, with many enduring long-term consequences and significant medical and rehabilitation costs. The primary injury causes physical damage to neurons, glia, fiber tracts and microvasculature, which is then followed by secondary injury, consisting of pathophysiological mechanisms including an immune response, inflammation, edema, excitotoxicity, oxidative damage, and cell death. Most attempts at intervention focus on protection, repair or regeneration, with regenerative medicine becoming an intensively studied area over the past decade. The use of stem cells has been studied in many disease and injury models, using stem cells from a variety of sources and applications. In this study, human adipose-derived mesenchymal stromal cells (MSCs) were administered at early (3 days) and delayed (14 days) time points after controlled cortical impact (CCI) injury in rats. Animals were routinely assessed for neurological and vestibulomotor deficits, and at 32 days post-injury, brain tissue was processed by flow cytometry and immunohistochemistry to analyze neuroinflammation. Treatment with HB-adMSC at either 3d or 14d after injury resulted in significant improvements in neurocognitive outcome and a change in neuroinflammation one month after injury.

Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application.

For several decades, multipotent mesenchymal stromal cells (MSCs) have been extensively studied for their therapeutic potential across a wide range of diseases. In the preclinical setting, MSCs demonstrate consistent ability to promote tissue healing, down-regulate excessive inflammation and improve outcomes in animal models. Several proposed mechanisms of action have been posited and demonstrated across an array of in vitro models. However, translation into clinical practice has proven considerably more difficult. A number of prominent well-funded late-phase clinical trials have failed, thus calling out for new efforts to optimize product delivery in the clinical setting. In this review, we discuss novel topics critical to the successful translation of MSCs from pre-clinical to clinical applications. In particular, we focus on the major routes of cell delivery, aspects related to hemocompatibility, and potential safety concerns associated with MSC therapy in the different settings.

Human umbilical cord blood cells restore vascular integrity in injured rat brain and modulate inflammation in vitro.

Aim: Traumatic brain injury is a complex condition consisting of a mechanical injury with neurovascular disruption and inflammation with limited clinical interventions available. A growing number of studies report systemic delivery of human umbilical cord blood (HUCB) as a therapy for neural injuries. Materials & methods: HUCB cells from five donors were tested to improve blood-brain barrier integrity in a traumatic brain injury rat model at a dose of 2.5 × 107 cells/kg at 24 or 72 h postinjury and for immunomodulatory activity in vitro. Results & Conclusion: We observed that cells delivered 72 h postinjury significantly restored blood-brain barrier integrity. HUCB cells reduced the amount of TNF-α and IFN-γ released by activated primary rat splenocytes, which correlated with the expression of COX2 and IDO1.

Pre-injury monocyte/macrophage depletion results in increased blood-brain barrier permeability after traumatic brain injury.

Traumatic brain injury (TBI) effects both the brain and the immune system. Circulating monocytes/macrophages (Mo /Ma ) after a TBI may play an important role in preserving the blood-brain barrier (BBB), reducing brain edema, and interacting with resident microglia. To elucidate the role of circulating Mo /Ma , we utilized a monocyte/macrophage depletion model in response to TBI in male rats. Clodronate liposomes (CL) were used to deplete circulating Mo /Ma . A controlled cortical impact (CCI) injury model was used to create a TBI. All animals received either CL or PBS liposomes (PL), 48 and 24 hr prior to the procedure, and were sacrificed 72 hr post-injury for analysis of BBB permeability, brain edema, whole blood (Mo /Ma and granulocytes), and/or microglial analysis. Animals undergoing Mo /Ma depletion with CL prior to CCI (CCI-CL) were found to have increased BBB permeability when compared to non-depleted CCI (CCI-PL) animals. At 72 hr following injury, Sham-CL maintained on average an 82% reduction in the whole blood monocytes when compared to Sham-PL (p < 0.001). Monocytes in the whole blood remained significantly lower in CCI-CL animals when compared to CCI-PL (p < 0.001). The number of granulocytes in the whole blood of CCI-CL animals was higher at 3 days when compared to CCI-PL (p < 0.022). Surprisingly, the depletion of Mo /Ma did not affect brain edema. However, the depletion of Mo /Ma did result in a significant decrease in microglia (CCI-CL vs. CCI-PL, p < 0.012). In conclusion, an intact Mo /Ma population is required to repair BBB integrity and microglial response following injury.

Alternative splicing links histone modifications to stem cell fate decision.

BACKGROUND: Understanding the embryonic stem cell (ESC) fate decision between self-renewal and proper differentiation is important for developmental biology and regenerative medicine. Attention has focused on mechanisms involving histone modifications, alternative pre-messenger RNA splicing, and cell-cycle progression. However, their intricate interrelations and joint contributions to ESC fate decision remain unclear. RESULTS: We analyze the transcriptomes and epigenomes of human ESC and five types of differentiated cells. We identify thousands of alternatively spliced exons and reveal their development and lineage-dependent characterizations. Several histone modifications show dynamic changes in alternatively spliced exons and three are strongly associated with 52.8% of alternative splicing events upon hESC differentiation. The histone modification-associated alternatively spliced genes predominantly function in G2/M phases and ATM/ATR-mediated DNA damage response pathway for cell differentiation, whereas other alternatively spliced genes are enriched in the G1 phase and pathways for self-renewal. These results imply a potential epigenetic mechanism by which some histone modifications contribute to ESC fate decision through the regulation of alternative splicing in specific pathways and cell-cycle genes. Supported by experimental validations and extended datasets from Roadmap/ENCODE projects, we exemplify this mechanism by a cell-cycle-related transcription factor, PBX1, which regulates the pluripotency regulatory network by binding to NANOG. We suggest that the isoform switch from PBX1a to PBX1b links H3K36me3 to hESC fate determination through the PSIP1/SRSF1 adaptor, which results in the exon skipping of PBX1. CONCLUSION: We reveal the mechanism by which alternative splicing links histone modifications to stem cell fate decision.

Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modify Microglial Response and Improve Clinical Outcomes in Experimental Spinal Cord Injury.

No current clinical intervention can alter the course of acute spinal cord injury (SCI), or appreciably improve neurological outcome. Mesenchymal stromal cells (MSCs) have been shown to modulate the injury sequelae of SCI largely via paracrine effects, although the mechanisms remain incompletely understood. One potential modality is through secretion of extracellular vesicles (EVs). In this study, we investigate whether systemic administration of EVs isolated from human MSCs (MSCEv) has the potential to be efficacious as an alternative to cell-based therapy for SCI. Additionally, we investigate whether EVs isolated from human MSCs stimulated with pro-inflammatory cytokines have enhanced anti-inflammatory effects when administered after SCI. Immunohistochemistry supported the quantitative analysis, demonstrating a diminished inflammatory response with apparent astrocyte and microglia disorganization in cord tissue up to 10 mm caudal to the injury site. Locomotor recovery scores showed significant improvement among animals treated with MSCEv. Significant increases in mechanical sensitivity threshold were observed in animals treated with EVs from either naïve MSC (MSCEvwt) or stimulated MSC (MSCEv+), with a statistically significant increase in threshold for MSCEv+-treated animals when compared to those that received MSCEvwt. In conclusion, these data show that treatment of acute SCI with extracellular vesicles derived from human MSCs attenuates neuroinflammation and improves functional recovery.

Inflammation-Stimulated Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Inflammation.

Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) have been proposed to be a key mechanistic link in the therapeutic efficacy of cells in response to cellular injuries through paracrine effects. We hypothesize that inflammatory stimulation of MSCs results in the release of EVs that have greater anti-inflammatory effects. The present study evaluates the immunomodulatory abilities of EVs derived from inflammation-stimulated and naive MSCs (MSCEv+ and MSCEv, respectively) isolated using a current Good Manufacturing Practice-compliant tangential flow filtration system. Detailed characterization of both EVs revealed differences in protein composition, cytokine profiles, and RNA content, despite similarities in size and expression of common surface markers. MSCEv+ further attenuated release of pro-inflammatory cytokines in vitro when compared to MSCEv, with a distinctly different pattern of EV-uptake by activated primary leukocyte subpopulations. The efficacy of EVs was partially attributed to COX2/PGE2 expression. The present study demonstrates that inflammatory stimulation of MSCs renders release of EVs that have enhanced anti-inflammatory properties partially due to COX2/PGE2 pathway alteration. Stem Cells 2018;36:79-90.

Prostaglandin E2 Indicates Therapeutic Efficacy of Mesenchymal Stem Cells in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury, a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage, safety, and relative ease to isolate and culture. However, there has been a wide range in efficacy reported using MSC clinically and in preclinical models, likely due to differences in cell preparations and a significant amount of donor variability. In this study, we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC, one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI, an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed, the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment, suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430.

Propranolol and Mesenchymal Stromal Cells Combine to Treat Traumatic Brain Injury.

UNLABELLED: More than 6.5 million patients are burdened by the physical, cognitive, and psychosocial deficits associated with traumatic brain injury (TBI) in the U.S. Despite extensive efforts to develop neuroprotective therapies for this devastating disorder, there have been no successful outcomes in human clinical trials to date. Retrospective studies have shown that ß-adrenergic receptor blockers, specifically propranolol, significantly decrease mortality of TBI through mechanisms not yet fully elucidated but are thought to counterbalance a hyperadrenergic state resulting from a TBI. Conversely, cellular therapies have been shown to improve long-term behavior following TBI, likely by reducing inflammation. Given the nonredundancy in their therapeutic mechanisms, we hypothesized that a combination of acute propranolol followed by mesenchymal stem cells (MSCs) isolated from human bone marrow would have additive effects in treating a rodent model of TBI. We have found that the treatments are well-tolerated individually and in combination with no adverse events. MSCs decrease BBB permeability at 96 hours after injury, inhibit a significant accumulation of activated microglia/macrophage in the thalamic region of the brain both short and long term, and enhance neurogenesis short term. Propranolol decreases edema and reduces the number of fully activated microglia at 7 days and the number of semiactivated microglia at 120 days. Combinatory treatment improved cognitive and memory functions 120 days following TBI. Therefore, the results here suggest a new, efficacious sequential treatment for TBI may be achieved using the ß-blocker propranolol followed by MSC treatment. SIGNIFICANCE: Despite continuous efforts, traumatic brain injury (TBI) remains the leading cause of death and disability worldwide in patients under the age of 44. In this study, an animal model of moderate-severe TBI was treated with an acute dose of propranolol followed by a delayed dose of human mesenchymal stem cells (MSCs), resulting in improved short- and long-term measurements. These results have direct translational application. They reinforce the inevitable clinical trial of MSCs to treat TBI by demonstrating, among other benefits, a notable decrease in chronic neuroinflammation. More importantly, these results demonstrate that MSCs and propranolol, which is increasingly being used clinically for TBI, are compatible treatments that improve overall outcome.

MSCs and hyaluronan: sticking together for new therapeutic potential?

Research involving mesenchymal multipotent/stem/progenitor/stromal/marrow cells (MSCs) have translated to clinical trials at an extraordinary pace. By the time of this review, the public clinical trials database (http://clinicaltrials.gov) has 394 clinical trials listed using MSCs for a very wide range of therapeutic applications. Unexpectedly, the explanation for the increase in clinical trials using MSCs does not lie on a well-defined therapeutic mechanism--dramatic results have been demonstrated in a variety of studies involving different animal models of diseases, often describing discrete therapeutic mechanisms exerted by MSCs. This review will focus on recent data suggesting the involvement of hyaluronic acid (HA) in the beneficial effects of MSCs, evaluate the potential of MSC as modulators of HA and the implications of this modulation for disease therapy.