Immunomodulatory potential of human clonal mesenchymal stem cells and their extracellular vesicle subpopulations in an inflammatory-mediated diabetic Rhesus monkey model.

AIMS: This study aimed to investigate the therapeutic potential of a homogenous clonal population of mesenchymal stem cells (cMSC) and their extracellular vesicles (cMSC-EV) subpopulations on isolated rat islets in vitro and in inflammatory-mediated type 1 diabetes (T1D) non-human primate models. MAIN METHODS: EV subpopulations were isolated from human bone marrow-derived cMSC supernatant by low- and high-speed ultracentrifuge (EV-20K and EV-U110K) and sucrose density gradient (EV-S110K). The EVs were characterized generally and for the level of albumin, acetylcholinesterase (AChE) activity, co-isolate apoptotic markers, and expression of CD63+/annexin V+. Rat islet-derived single cells (iSCs) proliferation was measured using a Ki-67 proliferation assay. Diabetes was induced by multiple low-dose administrations of streptozotocin in rhesus monkeys. The diabetic monkeys were divided into three groups: the cMSC group, received two injections of 1.5 × 106 cMSC/kg body weight; the EV group received two injections of EVs isolated from 1.5 × 106 cMSC/kg, and the vehicle group received phosphate-buffered saline. KEY FINDINGS: EV-S110K showed higher AChE activity, lower expression of CD63+/annexin V+, and lower apoptotic co-isolates. EV-S110K induced ß-cell proliferation in vitro in a dose-dependent manner. The administration of EV-S110K and/or cMSC in diabetic monkeys demonstrated no significant changes in general diabetic indices and ß-cell mass in the pancreas of the monkeys. Both treatments demonstrated a lowering trend in blood glucose levels and reduced pro-inflammatory cytokines. In contrast, regulatory T cells and anti-inflammatory cytokines were increased. SIGNIFICANCE: cMSC and cMSC-EV provided initial evidence to attenuate clinical symptoms in inflammatory-mediated T1D non-human primates through immunomodulation.

Comparison of Ultrasound-Guided Percutaneous and Open Surgery Approaches in The Animal Model of Tumor Necrosis Factor-Alpha-Induced Disc Degeneration.

OBJECTIVE: Animal models provide a deeper understanding about various complications and better demonstrate the effect of therapeutic approaches. One of the issues in the low back pain (LBP) model is the invasiveness of the procedure and it does not mimic actual disease conditions in humans. The purpose of the present study was to compare the ultrasound-guided (US-guided) percutaneous approach with the open-surgery method in the tumor necrosis factor-alpha (TNF-α)-induced disc degeneration model for the first time to showcase the advantages of this recently developed, minimally invasive method. MATERIALS AND METHODS: In this experimental study, eight male rabbits were divided into two groups (open-surgery and US-guided). Relevant discs were punctured by two approaches and TNF-α was injected into them. Magnetic resonance imaging (MRI) was performed to assess the disc height index (DHI) at all stages. Also morphological changes (annulus fibrosus, nucleus pulposus) were evaluated by assessing Pfirrmann grade and histological evaluation (Hematoxylin and Eosin). RESULTS: The findings indicated targeted discs became degenerated after six weeks. DHI in both groups was significantly reduced (P<0.0001), however the difference was not significant between the two groups. In the open-surgery group, osteophyte formation was seen at six and eighteen weeks after the puncture. Pfirrmann grading revealed significant differences between injured and adjacent uninjured discs (P<0.0001). The US-guided method indicated significantly fewer signs of degeneration after six (P=0.0110) and eighteen (P=0.0328) weeks. Histological scoring showed significantly lower degeneration in the US-guided group (P=0.0039). CONCLUSION: The US-guided method developed a milder grade condition and such a model better mimics the chronic characteristics of LBP and the procedure is more ethically accepted. Therefore, the US-guided method could be a merit approach for future research in this domain as a safe, practical and low-cost method.

Effect of Mechanical Micro-Vibrations on The Efficiency of Leopard Inter-Species Somatic Cell Nuclear Transfer.

OBJECTIVE: Scarcity of oocytes for assisted reproduction in endangered species can be bypassed by interspecies somatic cell nuclear transfer (iSCNT). In Felids, domestic cat (Felis catus) oocytes can serve as recipients for the nucleus of the endangered Persian leopard (Panthera pardus saxicolor). However, in vitro oocyte maturation is still suboptimal in cats, whereas it has been reported to benefit from micro-vibration in non-felid species. Therefore, the present study is aimed to determine whether micro-vibration, applied during in vitro maturation (IVM), improves the embryogenic potential of cat oocytes transplanted with fibroblast nuclei of the Persian leopard. MATERIALS AND METHODS: In the experimental study, cat cumulus-oocyte complexes (COCs) were randomly assigned to the treatment group (micro-vibration) or control group (static culture). Resultant metaphase II (MII) oocytes were enucleated and reconstructed with nucleus transplants from leopard fibroblasts, followed by artificial oocyte activation and embryo culture under the same condition (static) for 7 days. RESULTS: While cumulus cell expansion and oocyte maturation profited from micro-vibration (P<0.05), the quantity and quality of blastocysts were significantly lower in micro-vibration than in the control group (P<0.05). The total number of blastocyst cells tended to be lower in the micro-vibration than in the control group (P=0.075). Nevertheless, the proportion of ICM and TE cells did not differ between the micro-vibration and control groups (P>0.05). CONCLUSION: The present study indicated that micro-vibration at a frequency of 44 Hz for 5 secs per hour enhanced nuclear maturation and cumulus cell expansion of cat oocytes. However, exposure to micro-vibration during IVM impaired the survival rate of reconstructed oocytes during the iSCNT process and their developmental competence toward the blastocyst stage.

Standard toxicity study of clinical-grade allogeneic human bone marrow-derived clonal mesenchymal stromal cells.

INTRODUCTION: Mesenchymal stromal cells (MSCs) have opened a new window to treat inflammatory and non-inflammatory diseases. Nonetheless, their clinical applications require rigorous control and monitoring procedures to ensure full compliance with the principles of good manufacturing practice (GMP). Various evaluations should be passed in conjunction with the development of these newly emerging therapeutic products from bench-to-bedside. These evaluations include in vitro characterization, preclinical studies, and clinical trials to ensure product safety and efficacy. Therefore, a robust and well-designed preclinical study is critical to confirm product safety. This study aims to determine the probable toxicity effects of local and systemic injections of cryopreserved human bone marrow-derived clonal MSCs (BM-cMSCs) during subacute and subchronic periods of time. METHODS: BM-cMSCs were characterized according to the International Society for Cell and Gene Therapy (ISCT) criteria for MSCs. Both safety and toxicity of the BM-cMSCs population produced under GMP-compatible conditions were assessed in both sexes of Sprague Dawley (SD) rats via systemic intravenous (IV) administration and local injection in intervertebral disc (IVD). Behavioral changes, clinical signs of toxicity, and changes in body weight, water and food consumption were the important variables for product toxicity testing over 14 consecutive days during the subacute period and 90 consecutive days during the subchronic period. At the end of the assessment periods, the rats were killed for histopathology analysis of the target tissues. The BM-cMSCs potential for tumorigenicity was checked in nude mice. RESULTS: Single IV and IVD injections of BM-cMSCs did not cause significant signs of clinical toxicity, or changes in laboratory and histopathology data during the subacute (14 day) and subchronic (90 day) periods. Ex vivo-expanded and cryopreserved BM-cMSCs did not induce tumor formation in nude mice. CONCLUSION: The results suggest that local and systemic administrations of xenogeneic BM-cMSCs in both sexes of SD rats do not cause toxicity during the subacute and subchronic periods of time. Also, BM-cMSCs were non-tumorigenic in nude mice.

The Cross-Talks Among Bone Morphogenetic Protein (BMP) Signaling and Other Prominent Pathways Involved in Neural Differentiation.

The bone morphogenetic proteins (BMPs) are a group of potent morphogens which are critical for the patterning, development, and function of the central nervous system. The appropriate function of the BMP pathway depends on its interaction with other signaling pathways involved in neural differentiation, leading to synergistic or antagonistic effects and ultimately favorable biological outcomes. These opposite or cooperative effects are observed when BMP interacts with fibroblast growth factor (FGF), cytokines, Notch, Sonic Hedgehog (Shh), and Wnt pathways to regulate the impact of BMP-induced signaling in neural differentiation. Herein, we review the cross-talk between BMP signaling and the prominent signaling pathways involved in neural differentiation, emphasizing the underlying basic molecular mechanisms regarding the process of neural differentiation. Knowing these cross-talks can help us to develop new approaches in regenerative medicine and stem cell based therapy. Recently, cell therapy has received significant attention as a promising treatment for traumatic or neurodegenerative diseases. Therefore, it is important to know the signaling pathways involved in stem cell differentiation toward neural cells. Our better insight into the cross-talk of signaling pathways during neural development would improve neural differentiation within in vitro tissue engineering approaches and pre-clinical practices and develop futuristic therapeutic strategies for patients with neurological disease.

Macrophage polarization by MSC-derived CXCL12 determines tumor growth.

BACKGROUND: Phenotypic and functional heterogeneity of macrophages is known to be the main reason for their ability to regulate inflammation and promote tumorigenesis. Mesenchymal stem cells (MSCs) are one of the principal cells commonly found in the tumor stromal niche, with capability of macrophage phenotypic switching. The objective of this study was to evaluate the role of C-X-C motif chemokine ligand 12 (CXCL12) produced by marrow-derived MSCs in the phenotypic and functional pattern of bone marrow-derived macrophages (BMDMs). METHODS: First, the CRISPR/Cas9 system was used for the CXCL12 gene knock-out in MSCs. Then, coculture systems were used to investigate the role of MSCsCXCL12-/- and MSCsCXCL12+/+ in determination of macrophage phenotype. To further analyze the role of the MSC-derived CXCL12 niche, cocultures of 4T1 mammary tumor cells and macrophages primed with MSCsCXCL12-/- or MSCsCXCL12+/+ as well as in-vivo limiting dilution assays were performed. RESULTS: Our results revealed that the expression of IL-4, IL-10, TGF-ß and CD206 as M2 markers was significantly increased in macrophages co-cultured with MSCsCXCL12+/+ , whereas the expression of IL-6, TNF-α and iNOS was conversely decreased. The number and size of multicellular tumor spheroids were remarkably higher when 4T1 cells were cocultured with MSCCXCL12+/+-induced M2 macrophages. We also found that the occurrence of tumors was significantly higher in coinjection of 4T1 cells with MSCCXCL12+/+-primed macrophages. Tumor initiating cells were significantly decreased after coinjection of 4T1 cells with macrophages pretreated with MSCsCXCL12-/-. CONCLUSIONS: In conclusion, our findings shed new light on the role of MSC-derived CXCL12 in macrophage phenotypic switching to M2, affecting their function in tumorigenesis.

Immunomodulatory Activity of Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Prolongs Allogenic Skin Graft Survival in Nonhuman Primates.

OBJECTIVE: In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation. MATERIALS AND METHODS: In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H and E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses. RESULTS: A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05). CONCLUSION: It seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

Combined therapy of mesenchymal stem cells with a GLP-1 receptor agonist, liraglutide, on an inflammatory-mediated diabetic non-human primate model.

AIMS: Immunomodulation concurrent with the promotion of ß-cell function is a strategy used to develop innovative therapies for type 1 diabetes (T1D). Here, we assessed the therapeutic potential of co-administration of human clonal mesenchymal stem (stromal) cells (hBM-cMSCs) and liraglutide as a glucagon-like peptide-1 agonist in a non-human primate model with streptozotocin (STZ)-induced diabetes. MAIN METHODS: Diabetes was induced through intravenous (i.v.) multiple low-dose (MLD) infusions of STZ at a dose of 30 mg/kg body weight (b.w.) for five consecutive days, followed by two booster injections of 35 mg/kg on days 12 and 19. After 90 days, the diabetic animals were randomly allocated to two groups: The combination therapy group (n = 4) received injections of 1.5 × 106 hBM-cMSCs/kg b.w. through celiac artery by angiography on days 91 and 105 and daily subcutaneous injections of liraglutide (up to 1.8 mg/day) until day 160 while vehicle group received phosphate-buffered saline. The monkeys were assessed for functional, immunological, and histological analysis. KEY FINDINGS: The combined treatment group had continued reduction in FBG levels up to day 160, which was accompanied by increased b.w., C-peptide, and ß-cell function, and decreased HbA1c and fructosamine levels compared to vehicle group. The combined treatment increased Tregs, IL-4, IL-10, and TGF-ß1 and decreased IL-6 and IL-1ß. Stereological analysis of the pancreatic tissue exhibited more total volume of insulin-secreting islets in the combined treatment group compared to vehicle group. SIGNIFICANCE: Our findings demonstrated this combined treatment impaired the clinical symptoms of diabetes in this animal model through immunomodulation and ß-cell preservation.

Two case reports of unusual phimosis in rhesus monkeys (Macaca Mulatta).

Phimosis is the inability of the penis to protrude from the prepuce. In the present report, we present two cases of phimosis in two rhesus monkeys. Surgical enlargement of preputial orifice was performed for unrestricted movement of penis. The exact cause of this condition is unknown to us.

Ratio of the Bronchial Lumen to Pulmonary Artery Diameter in Rhesus Macaques (Macaca mulatta) without Clinical Pulmonary Disease.

In human medicine, CT is widely used to detect changes in bronchial luminal diameter. The diameter of the artery that runs adjacent to the bronchus does not change dramatically along the airway path, such that this artery can be used as a reference to detect changes in the bronchial luminal diameter. The bronchoarterial ratio is increasingly used in veterinary medicine for the detection of lower airway diseases in animals. The purpose of this study was to establish the bronchoarterial ratio in rhesus macaques. We used CT to evaluate 12 rhesus macaques (Macaca mulatta) without clinical signs of pulmonary diseases and measured the bronchoarterial ratio in the right and left superior, middle, inferior and cardiac lung lobes. The overall bronchoarterial ratio (mean ± 1 SD) at all 7 locations in the 12 macaques was 0.59 ± 0.05. Moreover, there was no correlation between the BA ratio and age or sex in the study population. However, the BA ratio and weight of animals showed positive linear correlation. In this study, we established the reference range for the bronchoarterial ratio in clinically healthy rhesus macaques. This ratio is consistent among lung lobes and between animals.

Role of basal stress hormones and amygdala dimensions in stress coping strategies of male rhesus monkeys in response to a hazard-reward conflict.

OBJECTIVES: In the present study the effect of stress on monkeys that had learned to retrieve food from a five-chamber receptacle, as well as the relationship between their behavior and the serum cortisol and epinephrine levels and relative size of the amygdala was evaluated. MATERIALS AND METHODS: Six male rhesus monkeys were individually given access to the food reward orderly. They could easily retrieve the rewards from all chambers except for the chamber 4, which a brief, mild electric shock (3 V) was delivered to them upon touching the chamber's interior. The coping behaviors were video-recorded and analyzed offline. Baseline serum cortisol and epinephrine levels were measured before the experiments using monkey enzyme-linked immunosorbent assay kit. One week after the behavioral experiment, the monkeys' brains were scanned using magnetic resonance imaging under general anesthesia. The cross-sectional area of the left amygdala in sagittal plane relative to the area of the whole brain in the same slice was evaluated by the planimetric method using ImageJ software. RESULTS: Exposure to the distressing condition caused different behavioral responses. Monkeys with higher baseline levels of serum cortisol and epinephrine and larger amygdala behaved more violently in the face of stress, indicating adopting emotion-focused stress-coping strategies. Conversely, those with low plasma epinephrine, moderate cortisol, and smaller amygdala showed perseverative behavior, indicating a problem-focused coping style. CONCLUSION: In dealing with the same stress, different responses might be observed from nonhuman primates according to their cortisol and epinephrine levels as well as their amygdala dimensions.

Autologous transplantation of mesenchymal stromal cells tends to prevent progress of interstitial fibrosis in a rhesus Macaca mulatta monkey model of chronic kidney disease.

BACKGROUND AIMS: Chronic kidney disease (CKD) attributed to cisplatin is well documented. Mesenchymal stromal cells (MSCs) are proven to be renotropic. Although they have been shown to improve function in CKD and reduce fibrosis in different experimental rodent models, their efficiency in primates is unknown. The present study aimed to evaluate the prevention of CKD and reduction of fibrosis in monkeys treated with MSCs after cisplatin nephrotoxicity. METHODS: We induced CKD in adult rhesus Macaca mulatta monkeys by means of intravenous administration of cisplatin. Autologous MSCs were transplanted by means of intrarenal arterial injections to assess the adverse effects of cisplatin in two CKD models: preventative and stable. Preventative CKD monkeys (n = 3) underwent cell transplantation 4 days after the cisplatin injection. The stable CKD monkeys (n = 2) underwent cell transplantation 6 months after the cisplatin injection. Non-treated (n = 4) and normal saline-injected animals (n = 3) comprised the control and vehicle groups, respectively. We followed the animals for survival rate, serum biochemistry, urine analysis and histopathological indices. RESULTS: In the preventive CKD model, MSC transplantation tended to improve some renal functions but significantly reduced the histopathologic score compared with the vehicle and control groups. In the stable CKD model, MSCs did not ameliorate renal function or pathological score. CONCLUSIONS: These results suggest that MSCs tend to delay progression of CKD and fibrosis but do not reduce established interstitial fibrosis in this unique primate model of cisplatin-induced nephrotoxicity.

Intra-renal arterial injection of autologous bone marrow mesenchymal stromal cells ameliorates cisplatin-induced acute kidney injury in a rhesus Macaque mulatta monkey model.

BACKGROUND: Clinically, acute kidney injury (AKI) is a potentially devastating condition for which no specific therapy improves efficacy of the repair process. Bone marrow mesenchymal stromal cells (BM-MSCs) are proven to be beneficial for the renal repair process after AKI in different experimental rodent models, but their efficacy in large animals and humans remains unknown. This study aims to assess the effect of autologous rhesus Macaque mulatta monkey BM-MSC transplantation in cisplatin-induced AKI. METHODS: We chose a model of AKI induced by intravenous administration of 5 mg/kg cisplatin. BM-MSCs were transplanted through intra-arterial injection. The animals were followed for survival, biochemistry analysis and pathology. RESULTS: Transplantation of 5 × 10(6) cells/kg ameliorated renal function during the first week, as shown by significantly lower serum creatinine and urea values and higher urine creatinine and urea clearance without hyponatremia, hyperkalemia, proteinuria and polyuria up to 84 d compared with the vehicle and control groups. The superparamagnetic iron oxide nanoparticle-labeled cells were found in both the glomeruli and tubules. BM-MSCs markedly accelerated Foxp3+ T-regulatory cells in response to cisplatin-induced damage, as revealed by higher numbers of Foxp3+ cells within the tubuli of these monkeys compared with cisplatin-treated monkeys in the control and vehicle groups. CONCLUSIONS: These data demonstrate that BM-MSCs in this unique large-animal model of cisplatin-induced AKI exhibited recovery and protective properties.

Transplantation of adult monkey neural stem cells into a contusion spinal cord injury model in rhesus macaque monkeys.

OBJECTIVE: Currently, cellular transplantation for spinal cord injuries (SCI) is the subject of numerous preclinical studies. Among the many cell types in the adult brain, there is a unique subpopulation of neural stem cells (NSC) that can self-renew and differentiate into neurons. The study aims, therefore, to explore the efficacy of adult monkey NSC (mNSC) in a primate SCI model. MATERIALS AND METHODS: In this experimental study, isolated mNSCs were analyzed by flow cytometry, immunocytochemistry, and RT-PCR. Next, BrdU-labeled cells were transplanted into a SCI model. The SCI animal model was confirmed by magnetic resonance imaging (MRI) and histological analysis. Animals were clinically observed for 6 months. RESULTS: Analysis confirmed homing of mNSCs into the injury site. Transplanted cells expressed neuronal markers (TubIII). Hind limb performance improved in trans- planted animals based on Tarlov's scale and our established behavioral tests for monkeys. CONCLUSION: Our findings have indicated that mNSCs can facilitate recovery in contusion SCI models in rhesus macaque monkeys. Additional studies are necessary to determine the im- provement mechanisms after cell transplantation.

Implantation of amniotic membrane as a vascular substitute in the external jugular vein of juvenile sheep.

OBJECTIVE: Amniotic membrane, as a natural biomaterial, has many advantages, such as low immunogenicity, anti-inflammation, antifibrosis, and rich extracellular matrix components, which make it a promising source for vascular tissue engineering. This study assessed the feasibility of constructing a vein conduit from the amniotic membrane and implanting it in the external jugular vein of juvenile sheep. METHODS: Human amniotic membrane was prepared using fresh human placenta. For construction of a tube such as a vein, the membrane was rolled around a tube and amniotic membrane-constructed conduits were interposed to the external jugular vein by end-to-end anastomosis. Grafts were assessed for patency at weeks 5 and 48 and explanted for evaluation with histologic and microscopic techniques. RESULTS: At 5 weeks after implantation, the grafts were completely patent and displayed no signs of dilation. The internal surface was smooth and shiny, without any evidence of thrombus formation. After 48 weeks, grafts were still completely patent and displayed no signs of intimal thickening, dilation, or stenosis. No inflammation or fibrosis was evident. Histologic evaluation of the explanted grafts demonstrated a monolayer of endothelial cells. Scanning electron microscopy revealed a confluent layer of cells with normal endothelial cell morphology. A monolayer of cells positive for von Willebrand factor was detected in histology sections. CONCLUSIONS: The findings of this study confirm that the amniotic membrane can be a proper substitute for vascular tissue engineering.