Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage

Background aimsStem cell therapy seems to be a promising therapeutic tool for treating central nervous system (CNS) injuries. Bone marrow stromal cell (BMSC) transplantation influences functional outcome subsequent to intracerebral hemorrhage (ICH), and enhances endogenous neurogenesis in acute condition studies. We investigated whether late administration of BMSC improves functional deficits subsequent to ICH.MethodsExperimental ICH was induced by stereotactic injection of 0.5 IU collagenase type IV in the striatum of adult female Wistar rats, and 2 months later intralesional administration of 5 × 10⁶ allogeneic BMSC from male donors rats in saline (n = 10), or saline only (n = 10), was performed. In the following 6 months, functional outcome was evaluated in each animal by rotarod, modified neurologic severity score (mNSS) and video-tracking box (VTB) tests. To study the behavior of BMSC after transplantation, in situ hybridization studies were performed, with double labeling of the chromosome Y-linked SrY-gene, and neuronal nuclei (NeuN) protein or gliofibrillary acidic protein (GFAP).ResultsThe assessment test revealed significant improvements in functional outcome for the BMSC-treated animals after 2 months of follow-up. Histologic results showed that functional outcome was associated with strong reactivation of endogenous neurogenesis. Furthermore, intralesional BMSC not only integrated in the injured tissue but also showed phenotypic expression of GFAP and NeuN.ConclusionsLate intracerebral transplantation of allogeneic BMSC induces functional recovery after ICH. The possibility of using this type of cell therapy to reverse the consequences of hemorrhagic stroke in humans should be considered.     

Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds

Background aimsCell therapy using bone marrow stromal cells (BMSCs) has been considered a promising strategy for neurologic sequelae after intracerebral hemorrhage (ICH). However, after intracerebral administration of BMSCs, most of the cells die, partly because of the absence of extracellular matrix. Intracerebral transplantation of BMSCs, supported in a platelet-rich plasma (PRP) scaffold, optimizes this type of cell therapy.MethodsICH was induced by stereotactic injection of 0.5 IU of collagenase type IV in the striatum of adult Wistar rats (n = 40). Two months later, the rats were subjected to intracerebral administration of 5 × 10⁶ allogeneic BMSCs embedded in a PRP scaffold (n = 10), 5 × 10⁶ allogeneic BMSCs in saline (n = 10), PRP-derived scaffold only (n = 10) or saline only (n = 10). Functional improvements in each group over the next 6 months were assessed using Rotarod and Video-Tracking-Box tests. Endogenous neurogenesis and survival of transplanted BMSCs were examined at the end of follow-up.ResultsOur study demonstrated neurologic improvement after BMSC transplantation and significantly better functional improvement for the group of animals that received BMSCs in the PRP-derived scaffold compared with the group that received BMSCs in saline. Histologic results showed that better functional outcome was associated with strong activation of endogenous neurogenesis. After intracerebral administration of BMSCs, donor cells were integrated in the injured tissue and showed phenotypic expression of glial fibrillary acidic protein and neuronal nucleus.ConclusionsPRP-derived scaffolds increase the viability and biologic activity of BMSCs and optimize functional recovery when this type of cell therapy is applied after ICH.     

Bone marrow stromal cell transplantation combined with angiotensin-converting enzyme inhibitor treatment in rat with acute myocardial infarction and the role of insulin-like growth factor-1

Background aimsWe investigated bone marrow stromal cell (BMSC) transplantation combined with angiotensin-converting enzyme inhibitor (ACEI) treatment in acute myocardial infarction (AMI) and the role of insulin-like growth factor-1 (IGF-1)MethodsAMI models were established in Sprague–Dawley rats by ligation of the left anterior descending coronary artery and grouped into blank control (BC), ACEI treatment (ACEI), BMSC transplantation (BMSC) and BMSC transplantation plus ACEI (combined). Perindopril (2.5 mg/kg) was administered by gavage to ACEI and combined groups from the day after AMI. BMSC (2 × 10⁸) were injected into the border of the MI area a week later in the BMSC and combined groups.ResultsAfter 4 weeks, hemodynamics in the BMSC and combined groups were significantly improved (P < 0.05 versus BC), with the greatest improvement in the combined group (P < 0.05). In addition, an increased number of BMSC survived in the combined group (P < 0.05 versus BMSC). A proportion of BMSC was positive for troponin T, as detected by immunofluorescence. The number of apoptotic cardiomyocytes was decreased in the BMSC and ACEI groups, and even further in the combined group (P < 0.05). IGF-1 expression was up-regulated in the BMSC and combined groups (P < 0.05 versus BC), but not in the ACEI group. B cell lymphoma-2 (Bcl-2) expression was up-regulated in the ACEI, BMSC and combined groups, with the highest expression in the combined group (P < 0.05).ConclusionsOur results show that BMSC engrafted in AMI can survive well and secrete IGF-1 and preserve cardiac function significantly. These data suggest that BMSC transplantation inhibits apoptosis of cardiomyocytes by up-regulation of Bcl-2 expression in the myocardium, and this effect might be sensitized by ACEI.     

CXCR4 and matrix metalloproteinase-2 are involved in mesenchymal stromal cell homing and engraftment to tumors

Background aimsBone marrow-derived mesenchymal stromal cells (BMSC) have been shown to migrate to injury, ischemia and tumor microenvironments. The mechanisms by which mesenchymal stromal cells (MSC) migrate across endothelium and home to the target tissues are not yet fully understood.MethodsWe used rat BMSC to investigate the molecular mechanisms involved in their tropism to tumors in vitro and in vivo.ResultsBMSC were shown to migrate toward four different tumor cells in vitro, and home to both subcutaneous and lung metastatic prostate tumor models in vivo. Gene expression profiles of MSC exposed to conditioned medium (CM) of various tumor cells were compared and revealed that matrix metalloproteinase-2 (MMP-2) expression in BMSC was downregulated after 24 h exposure to tumor CM. Chemokine (C–X–C motif) Receptor 4 (CXCR4) upregulation was also found in BMSC after 24 h exposure to tumor CM. Exposure to tumor cell CM enhanced migration of BMSC toward tumor cells. Stromal Cell-Derived Factor (SDF-1) inhibitor AMD3100 and MMP-2 inhibitor partly abolished the BMSC migration toward tumor cells in vitro.ConclusionsThese results suggest that the CXCR4 and MMP-2 are involved in the multistep migration processes of BMSC tropism to tumors.     

Influence of related donor age on outcomes after peripheral blood stem cell transplantation

Background aimsThe rising use of allogeneic transplantation in older recipients necessitates considering older related donors. The effect of related donor age for peripheral blood stem cell allografts (PBSC) on graft maintenance and outcomes, independent of CD34⁺cell dose, has not been well-characterized.MethodsHLA-related donors (98% siblings) underwent a uniform filgrastim-based mobilization regimen aiming to collect and infuse 5 × 10⁶ CD34⁺ cells/recipient kg. Donor and recipient age were modeled in multiple ways to account for the correlation, and outcomes reported by decade of donor age.ResultsThe median donor and recipient ages were 52 years and 54 years, respectively. The mean CD34⁺ cell dose infused was 5.6 × 10⁶ CD34⁺/kg and 75% of patients received a narrow range between 4.4 and 6.6 × 10⁶ CD34⁺ cells/kg. Neither better PBSC mobilization nor higher CD34⁺ content of allografts was significantly associated with engraftment or transplant outcomes. After adjusting for recipient age and other prognostic factors, older donor age by decade conferred a lower risk of non-relapse mortality (NRM) [hazard ratio (HR) = 0.64, 95% confidence interval (CI) 0.45–0.91, P = 0.013] and borderline improvement in overall survival (OS) (HR = 0.76, 95% CI 0.58–0.99, P = 0.045) without altering progression-free survival (PFS) (HR = 0.85, 95% CI 0.66–1.07, P = 0.18).ConclusionsOlder donor age does not worsen outcome after matched related donor PBSC transplantation in patients receiving a narrow range CD34⁺ cells. The relatively small sample size mandates that the finding of similar to improved outcomes for older related donor age must be confirmed in larger studies.     

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses

Background aimsThe development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horsesMethodsSixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injectionResultsMSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSCConclusionsThe healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.     

Cell transplantation for myocardial injury: a preliminary comparative study

Background aimsCell transplantation may restore viable muscle after myocardial infarction. Because many studies have focused on one cell type, we compared the characteristics of skeletal myoblasts (SKM), bone marrow stromal/stem cells (BMSC) and smooth muscle cells (SMC) and their effects on cardiac function after myocardial injury.MethodsIn vitro cell characteristics, including proliferation, hypoxic survival and vascular endothelial cell growth factor (VEGF) expression, of SKM, BMSC and SMC were compared. An in vivo left anterior descending artery ligation rat model was used, and cells were implanted into the infarct (n = 16 per cell type). Cell survival was determined by PKH26 staining and real-time polymerase chain reaction (PCR). Cardiac function, tissue VEGF and stem cell factor (SCF) expression and vasculogenesis were evaluated.ResultsAlthough cell morphologies were distinct, in vitro proliferation was similar. In vitro studies showed that SKM had the highest hypoxic survival, whereas BMSC had the lowest hypoxic survival but the highest VEGF expression. After implantation, SKM showed the highest overall survival and in vivo SCF expression, and both SMC and SKM expressed the highest VEGF levels. Vasculogenesis was significantly (P < 0.001) improved after transplantation of each cell type. Overall, BMSC and SKM promoted the greatest improvement in cardiac function.ConclusionsSKM, BMSC and SMC expressed VEGF and SCF and promoted vasculogenesis. Although BMSC showed the greatest regenerative potential relative to cell survival and growth factor expression, the greatest improvement in cardiac function was observed with BMSC and SKM.     

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.     

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy

Background aimsTransplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model.MethodsASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10⁵ cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed.ResultsASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group.ConclusionsCompared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.     

Stem cells from human exfoliated deciduous teeth (SHED) enhance wound healing and the possibility of novel cell therapy

Background aimsIn recent years, stem cells from human exfoliated deciduous teeth (SHED) have received attention as a novel stem cell source with multipotent potential. We examined the effect on wound-healing promotion with unique stem cells from deciduous teeth as a medical waste.MethodsAn excisional wound-splinting mouse model was used and the effect of wound healing among SHED, human mesenchymal stromal cells (hMSCs), human fibroblasts (hFibro) and a control (phosphate-buffered saline; PBS) was evaluated by macroscopy, histology and enzyme-linked immunosorbent assay (ELISA), and the expression of hyaluronan (HA), which is related to wound healing, investigated.ResultsSHED and hMSCs accelerated wound healing compared with hFibro and the control. There was a statistically significant difference in wound healing area among hFibro, hMSCs and SHED compared with the control after day 5. At days 7 and 14 after cell transplantation, the histologic observation showed that transplanted PKH26-positive cells were surrounded by human HA binding protein, especially in hMSCs and SHED. HA expression volume values were 1558.41 ± 60.33 (control), 2092.75 ± 42.56 (hFibro), 2342.07 ± 188.10 (hMSCs) and 2314.85 ± 164.91 (SHED) ng/mg, respectively, and significantly higher in hMSCs and SHED compared with hFibro and control at days 7 and 14 (P < 0.05).ConclusionsOur results show that SHED hMSCs have similar effects of wound-healing promotion as hFibro and controls. This implies that SHED might offer a unique stem cell resource and the possibility of novel cell therapies for wound healing in the future.     

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats

Background aimsBone marrow stromal cells (BMSC) have been shown to provide neuroprotection after transplantation into the injured central nervous system. The present study investigated whether adult rat BMSC differentiated along a Schwann cell lineage could increase production of trophic factors and support neuronal survival and axonal regeneration after transplantation into the injured spinal cord.MethodsAfter cervical C4 hemi-section, 5-bromo-2-deoxyuridine (BrdU)/green fluorescent protein (GFP)-labeled BMSC were injected into the lateral funiculus at 1 mm rostral and caudal to the lesion site. Spinal cords were analyzed 2–13 weeks after transplantation.Results and ConclusionsTreatment of native BMSC with Schwann cell-differentiating factors significantly increased production of brain-derived neurotrophic factor in vitro. Transplanted undifferentiated and differentiated BMSC remained at the injection sites, and in the trauma zone were often associated with neurofilament-positive fibers and increased levels of vascular endothelial growth factor. BMSC promoted extensive in-growth of serotonin-positive raphaespinal axons and calcitonin gene-related peptide (CGRP)-positive dorsal root sensory axons into the trauma zone, and significantly attenuated astroglial and microglial cell reactions, but induced aberrant sprouting of CGRP-immunoreactive axons in Rexed's lamina III. Differentiated BMSC provided neuroprotection for axotomized rubrospinal neurons and increased the density of rubrospinal axons in the dorsolateral funiculus rostral to the injury site. The present results suggest that BMSC induced along the Schwann cell lineage increase expression of trophic factors and have neuroprotective and growth-promoting effects after spinal cord injury.     

A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies

Background aimsRetroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose.MethodsWe tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia.ResultsMedian anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24–48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10⁸, n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia.ConclusionsThe method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.     

Clinical-grade varicella zoster virus-specific T cells produced for adoptive immunotherapy in hemopoietic stem cell transplant recipients

Background aimsVaricella zoster virus (VZV) causes life-long latent infection in healthy individuals, which reactivates in 10–68% of stem cell transplant patients. Reconstituting immunity through adoptive transfer of T cells specific for VZV may aid in the prophylaxis and treatment of VZV infections. The potential for generating T cells specific for VZV using a clinically approved VZV vaccine strain was investigated.MethodsThe Varivax^® vaccine was used to stimulate peripheral blood mononuclear cells from healthy donors. Only reagents approved for clinical manufacture were used. Monocyte-derived dendritic cells pulsed with Varivax (R) were used to stimulate autologous mononuclear cells at a responder to stimulator ratio of 10:1. On day 7, a second stimulation was performed; 20 U/mL interleukin (IL)-2 were added from day 7 and 50 U/mL IL-2 from day 14 onwards. Cell phenotype and functionality were assessed after 21 days of culture.ResultsA mean increase of 11-fold in cell number was observed (n = 18). Cultures were mainly T cells (mean CD3 ⁺ 89.7%, CD4 ⁺ 54.2%, CD8 ⁺ 28.7%) with effector and central memory phenotypes. Cells produced one or more T helper (Th)1 cytokine (interferon-γ, tumor necrosis factor-α and IL-2), and CD4 ⁺ (but not CD8 ⁺) cells expressed the cytoxicity marker CD107 when restimulated with VZV antigens.ConclusionsWe have demonstrated a clinically applicable method that yields high numbers of highly reactive T cells specific for VZV. We propose that reconstructing host immunity through adoptive transfer of VZV-specific T cells will reduce the frequency of clinical VZV infection in the period of severe immune suppression that follows allogeneic stem cell transplantation.     

Lymphocyte subset analysis for the assessment of treatment-related complications after autologous stem cell transplantation in multiple myeloma

Background aimsThe aim of this study was to investigate the correlation between infused lymphocyte populations and lymphocyte subsets at engraftment, and the early clinical implications of lymphocyte subset recovery after autologous stem cell transplantation (ASCT) in multiple myeloma (MM).MethodsWe examined the lymphocyte populations of infused autografts and the lymphocyte subsets of peripheral blood at engraftment from 50 patients using flow cytometry. Each subset was grouped as low (below median) and high (above median) to examine the correlation with mucositis of grade 3 or more and the occurrence of infections and cytomegalovirus (CMV) reactivation.ResultsUsing Spearman correlation coefficients, we found that cell doses of infused CD8⁺ (P = 0.042) and CD19⁺ cells (P = 0.044) were significantly associated with the absolute lymphocyte count (ALC) at engraftment. The dose of infused CD34⁺ cells was not associated with the change of lymphocyte subsets except for an inverse correlation with CD4⁺ cells (P = 0.006). After adjusting for potential variables in univariate analysis, multivariate analyzes revealed that the lower ratio of infused CD4⁺ to CD8⁺ cells (P = 0.030) was an independent factor for severe mucositis. Of lymphocyte subsets at engraftment, a higher frequency of CD3⁺ (P = 0.024) and a lower frequency of CD56⁺ (P = 0.020) were independent predictors for infections after engraftment. A higher frequency of CD8⁺ cells (P = 0.041) and a lower ratio of CD4⁺ to CD8⁺ (P = 0.021) were independent predictors for CMV reactivation.ConclusionsOur data suggest that lymphocyte subset analysis of infused autograft and peripheral blood at engraftment may provide new predictors for early complications after ASCT in patient with MM.     

Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate

Background aimsMesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC).MethodsSections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3).ResultsWe isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10¹⁰ cells (ranging from 1.0 × 10¹⁰ to 29.0 × 10¹⁰). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo.ConclusionsUC constitute a convenient and very rich source of MSC for the production of third-party ‘clinical doses’ of cells under good manufacturing practice (GMP) conditions.     

Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory CD4+CD25+ T lymphocytes in the peripheral blood of patients with advanced renal carcinoma

ObjectiveTo analyze the effect of Argon-Helium cryosurgery (AHCS) combined with transcatheter renal arterial embolization (TRAE) on the differentiation of regulatory CD4⁺ CD25⁺ T cell (Treg) and its implication in patients with renal carcinoma.MethodsSeventy seven patients are included in the study, and divided into two groups: TRAE group (n = 45, receiving TRAE only) and TRAE + cryoablation group (n = 32, receiving cryoablation 2–3 weeks after TRAE). The percentage of Treg cells and T lymphocyte subsets (CD4⁺T, CD8⁺T, and CD4⁺T/CD8⁺T) in the peripheral blood is measured by flow cytometry previous to the therapy and 3 months after therapy. Meanwhile, the extent of tumor necrosis is measured by MRI or CT 1 month after therapy.ResultsThe percentages of Treg cells of patients in TRAE + cryoablation group decrease from (6.65 ± 1.22)% to (3.93 ± 1.16)%, (t = 42.768, P < 0.01), and the percentages of CD4⁺T and CD4⁺T/CD8⁺T increase significantly (P < 0.01). However, the results of patients in TRAE group show that the percentages of Treg, CD4⁺T, CD8⁺T and CD4⁺T/CD8⁺T increase slightly although the differences had no statistical significance (P > 0.05). The tumor necrosis rate of TRAE + cryoablation group is 57.5%, significantly higher than those of TRAE group, which shows 31.6% (t = 6.784, P < 0.01). The median survival duration of the TRAE + cryoablation group is 20 months, significantly longer than that of the TRAE group (χ² = 7.368, P < 0.01). The decreasing extent of Treg cells is correlated with tumor necrosis rates (r = 0.90, P < 0.01) and life time (r = 0.67, P < 0.01).ConclusionThe therapy of TRAE combined with cryoablation contributes to reduce the percentage of Treg cells and improve the immune situation of patients with renal cell carcinoma, which consequently increase tumor necrosis rate and prolong the patients‘ survival duration.     

Intraperitoneal kisspeptin-10 administration induces dose-dependent degenerative changes in maturing rat testes

AimsKisspeptin, a peptide secreted by hypothalamic neurons, is a critical regulator of reproduction and puberty but its role in the regulation of gonadal maturation in sexually immature males is elusive. The present study investigated the effects of 12 days of pulsatile kisspeptin administration on gonadotropins and testosterone release and maturation of immature male gonads.Main methodsKisspeptin-10 was administered intraperitoneally at different dosage concentrations (1 μg, 1 ng, and 10 pg) to 5 weeks old prepubertal male rats, twice daily for 12 days. Plasma LH, FSH and testosterone concentrations were measured through competitive-binding radioimmunoassay. Spermatogenesis was studied mainly at stage VII of the spermatogenic cycle through light and electron microscopy.Key findingsAt the end of the treatments plasma LH and testosterone concentrations were reduced significantly at 1 ng and 1 μg kisspeptin doses (P < 0.05; P < 0.01). Type A spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, step 7 spermatids, elongated spermatids and daily sperm production decreased significantly (P < 0.05). Sertoli cell efficiency and total support capacity of Sertoli cells were reduced at all doses (P < 0.05). Meiotic index decreased (P < 0.05) at 1 μg dose only, whereas coefficient of mitosis increased at 1 ng and 1 μg (P < 0.01) kisspeptin doses. Histologically, degeneration of seminiferous tubules was evident showing tubular necrosis, multinucleated giant cell formation, intratubular vacuolization, widened lumen and deshaped germ cells. Marked ultrastructural changes characterized by thin basal laminae, enlarged intratubular spaces, abnormal acrosome and disrupted germ cells were noticeable.SignificanceIn conclusion long-term kisspeptin-10 administration negatively regulates gonadal maturation in prepubertal testes.     

Biphasic effect of EGb761 on simulated ischemia-induced rat BMSC survival in vitro and in vivo

AimsThe standardized extract from the leaves of Ginkgo biloba (EGb761) is applied as a phyto-pharmacon in therapy of diverse cardiovascular disorders. However, the effects of EGb761 on bone-marrow mesenchymal stem cells (BMSCs) transplanted into the ischemic myocardium currently remain uncertain. In this study, the dosage-effects of EGb761 on BMSC survival in vitro and in vivo were investigated.Main methodsThe ischemic microenvironment of rat BMSCs was simulated by hypoxia/serum deprivation (SD) and the rat myocardial infarction model was established. The rat BMSCs were cultured under hypoxia/SD or transplanted into the animal ischemic heart. The BMSC apoptosis was determined by FACS and TUNEL assay. Each apoptotic signal molecule's activity was assayed by immunoblot.Key findingsEGb761 showed a biphasic effect on the hypoxia/SD-induced BMSC apoptosis. Low concentration of EGb761 (10–100 μg/ml) aggravated hypoxia/SD-induced apoptosis via Akt inactivation and an enhancement of caspase-9 and caspase-3 expressions, whereas high concentration of EGb761 (500–2000 μg/ml) significantly prevented hypoxia/SD-induced BMSC apoptosis via the activated Akt and the inactivated caspase-9 and caspase-3. The animal study also indicated that the apoptotic index (AI) in the high concentration of EGb761 group was significantly lower than the low concentration of EGb761 group.SignificanceThe biphasic effect of EGb761 is closely related to the PI3K-Akt and caspase-9 signaling pathways. The therapeutic concentration of EGb761 may be one of the vital factors determining the specific action of EGb761 on cell apoptosis. It is of significant clinical implication to investigate the mechanisms of the biphasic effect of EGb761.