Early-phase administration of human amnion-derived stem cells ameliorates neurobehavioral deficits of intracerebral hemorrhage by suppressing local inflammation and apoptosis.

BACKGROUND: Intracerebral hemorrhage (ICH) is a significant cause of death and disabilities. Recently, cell therapies using mesenchymal stem cells have been shown to improve ICH-induced neurobehavioral deficits. Based on these findings, we designed this study to evaluate the therapeutic efficacy and underlying mechanisms by which human amnion-derived stem cells (hAMSCs) would ameliorate neurobehavioral deficits of ICH-bearing hosts. METHODS: hAMSCs were induced from amnia obtained by cesarean section and administered intravenously to ICH-bearing mice during the acute phase. The mice were then subject to multitask neurobehavioral tests at the subacute phase. We attempted to optimize the dosage and timing of the hAMSC administrations. In parallel with the hAMSCs, a tenfold higher dose of human adipose-derived stem cells (ADSCs) were used as an experimental control. Specimens were obtained from the ICH lesions to conduct immunostaining, flow cytometry, and Western blotting to elucidate the underlying mechanisms of the hAMSC treatment. RESULTS: The intravenous administration of hAMSCs to the ICH-bearing mice effectively improved their neurobehavioral deficits, particularly when the treatment was initiated at Day 1 after the ICH induction. Of note, the hAMSCs promoted clinical efficacy equivalent to or better than that of hADSCs at 1/10 the cell number. The systemically administered hAMSCs were found in the ICH lesions along with the local accumulation of macrophages/microglia. In detail, the hAMSC treatment decreased the number of CD11b+CD45+ and Ly6G+ cells in the ICH lesions, while splenocytes were not affected. Moreover, the hAMSC treatment decreased the number of apoptotic cells in the ICH lesions. These results were associated with suppression of the protein expression levels of macrophage-related factors iNOS and TNFα. CONCLUSIONS: Intravenous hAMSC administration during the acute phase would improve ICH-induced neurobehavioral disorders. The underlying mechanism was suggested to be the suppression of subacute inflammation and apoptosis by suppressing macrophage/microglia cell numbers and macrophage functions (such as TNFα and iNOS). From a clinical point of view, hAMSC-based treatment may be a novel strategy for the treatment of ICH.

Comparison of cryoprotectants in hematopoietic cell infusion-related adverse events.

BACKGROUND: The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated. STUDY DESIGN AND METHODS: To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration. RESULTS: The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats. CONCLUSIONS: Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

[Cell therapy using amnion-derived mesenchymal stem cells].

This study focused on amnion-derived mesenchymal stem cells (MSCs), which have immune- and inflammation-regulating properties, 1) a large number of stem cells, 2) high proliferative potential, and 3) are non-invasive to harvest. Based on the general research reported in many immune- and inflammation-related disease models, research on their commercial and therapeutic application was conducted. We have successfully manufactured a clinical trial product of amnion MSCs for the first time worldwide (clinical trial product name: AM01) and conducted physician-led clinical trials for acute graft versus host disease and Crohn's disease. Furthermore, CTEX Corporation, the first certified venture from Hyogo College of Medicine, was launched to further accelerate the clinical trial progression to obtain the manufacturing and marketing approval for amnion MSC AM01 to be used as a regenerative medical product at early stage.

Impact of the use of hydroxyethyl starch in granulocyte apheresis using Spectra Optia.

OBJECTIVES: To determine the impact of the use of hydroxyethyl starch (HES) in granulocyte apheresis using Spectra Optia. BACKGROUND: Granulocyte transfusion (GT) is a therapeutic option for neutropenic patients with severe bacterial or fungal infections. Recent studies in emergency medicine have shown the potential risk of using HES, which is routinely used in granulocyte apheresis to increase yield by sedimenting red blood cells. We hypothesized that the use of a newer device (Spectra Optia) would spare the need for HES. METHODS: We retrospectively compared granulocyte apheresis with HES (HES group, n = 89) and without HES (non-HES group, n = 36) using Spectra Optia. RESULTS: The granulocyte yield was significantly higher in the HES group (7.3 × 1010 vs. 2.0 × 10, p < 0.01) and was attributed to the difference in collection efficiency (36% vs. 7.7%, p < 0.01). The absolute neutrophil count on the following morning of GT was significantly higher in the HES group than in the non-HES group (2460/µl vs. 505/µl, p < 0.01). There were no significant differences in the occurrence of adverse events between the HES and non-HES groups. The renal function was unchanged in both groups after apheresis. CONCLUSIONS: We demonstrated that the advantage of using HES remained unchanged in granulocyte apheresis using Spectra Optia.

Preventive effect of mesenchymal stem cell culture supernatant on luminal stricture after endoscopic submucosal dissection in the rectum of pigs.

BACKGROUND: Mesenchymal stem cells (MSCs) are valuable in regenerative medicine, and MSC culture supernatant (MSC-CS) reportedly inhibits inflammation and fibrosis. We investigated whether colorectal luminal stricture develops after circumferential endoscopic submucosal dissection (ESD) in the colorectum, and whether the development of luminal stricture could be prevented by using MSC-CS enema. METHODS: In the first experiment, we performed circumferential ESD in the rectums or distal colons of pigs (n = 4 in each group). We sacrificed the pigs on Day 22 and measured the degree of luminal stricture. In the second experiment, we performed circumferential ESD in the rectums of pigs and administered an MSC-CS gel or a control gel enema after ESD for 4 days. We sacrificed the pigs on Day 8 (n = 3 in each group) or 22 (n = 3 in each group) to measure the degree of luminal stricture, and performed histological analysis. RESULTS: Severe luminal stricture was observed in the rectum but not in the distal colon. Moreover, fiber accumulation in the submucosa and hypertrophy of the muscularis propria were observed in the rectum but not in the distal colon. The degree of luminal stricture in the rectum was significantly lower in the MSC-CS group than in the control group. Furthermore, MSC-CS attenuated myofibroblast activation and hypertrophy of the muscularis propria on Day 22, and reduced inflammatory cell infiltration on Day 8. CONCLUSIONS: Luminal stricture after ESD developed only in the rectum because of the difference in myofibroblast activation and fiber accumulation. In addition, MSC-CS enema prevented luminal stricture after ESD, possibly by inhibiting the inflammatory reaction and fibrosis.

Oral administration of conditioned medium obtained from mesenchymal stem cell culture prevents subsequent stricture formation after esophageal submucosal dissection in pigs.

BACKGROUND AND AIMS: Endoscopic submucosal dissection (ESD) for esophageal cancer often causes postoperative stricture when more than three fourths of the circumference of the esophagus is dissected. Mesenchymal stem cells are a valuable cell source in regenerative medicine, and conditioned medium (CM) obtained from mesenchymal stem cells reportedly inhibits inflammation. In this study we evaluated whether CM could prevent esophageal stricture after ESD. METHODS: We resected a semi-circumference of pig esophagus by ESD. We prepared CM gel by mixing with 5% carboxymethyl cellulose and endoscopically applied it onto the wound bed immediately after ESD and on days 8 and 15 (weekly CM group) or administered it orally from days 1 to 4 (daily CM group). We also injected triamcinolone acetonide into the remaining submucosa immediately after ESD (steroid group). We killed the pigs on day 8 or day 22 to measure the stricture rate and to perform histologic analysis. RESULTS: Stricture rate in weekly and daily CM groups and steroid groups were significantly lower than in the control group on day 22. Moreover, CM significantly attenuated the number of activated myofibroblasts and fiber thickness on day 22. CM also significantly decreased the infiltration of neutrophils and macrophages compared with the control group on day 8. CONCLUSIONS: CM gel prevents esophageal stricture formation by suppressing myofibroblast activation and fibrosis after the infiltration of neutrophils and macrophages. Oral administration of CM gel is a promising treatment for the prevention of post-ESD stricture.

A novel mouse model of subcortical infarcts with dementia.

Subcortical white matter (WM) is a frequent target of ischemic injury and extensive WM lesions are important substrates of vascular cognitive impairment (VCI) in humans. However, ischemic stroke rodent models have been shown to mainly induce cerebral infarcts in the gray matter, while cerebral hypoperfusion models show only WM rarefaction without infarcts. The lack of animal models consistently replicating WM infarct damage may partially explain why many neuroprotective drugs for ischemic stroke or VCI have failed clinically, despite earlier success in preclinical experiments. Here, we report a novel animal model of WM infarct damage with cognitive impairment can be generated by surgical implantation of different devices to the right and left common carotid artery (CCA) in C57BL/6J mice. Implantation of an ameroid constrictor to the right CCA resulted in gradual occlusion of the vessel over 28 d, whereas placement of a microcoil to the left CCA induced ∼50% arterial stenosis. Arterial spin labeling showed a gradual reduction of cerebral blood flow over 28 d post operation. Such reductions were more marked in the right, compared with the left, hemisphere and in subcortical, rather than the cortical, areas. Histopathological analysis showed multiple infarct damage in right subcortical regions, including the corpus callosum, internal capsule, hippocampal fimbria, and caudoputamen, in 81% of mice. Mice displaying such damage performed significantly poorer in locomotor and cognitive tests. The current mouse model replicates the phenotypes of human subcortical VCI, including multiple WM infarcts with motor and cognitive impairment.

Evaluations of Intravenous Administration of CD34+ Human Umbilical Cord Blood Cells in a Mouse Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Several cell therapies have been explored as novel therapeutic strategies for neonatal encephalopathy because the benefits of current treatments are limited. We previously reported that intravenous administration of human umbilical cord blood (hUCB) CD34+ cells (hematopoietic stem cells/endothelial progenitor cells) at 48 h after insult exerts therapeutic effects in neonatal mice with stroke, i.e., permanent middle cerebral artery occlusion. Although neonatal stroke and hypoxic-ischemic encephalopathy (HIE) are grouped under the term "neonatal encephalopathy," their pathogenesis differs. However, little is known about the differences in the effects of the same treatment between these 2 diseases. In this study, we investigated whether the same treatment protocol exerts therapeutic effects in neonatal mice with HIE. The treatment significantly ameliorated the decreased cerebral blood flow in the ischemic penumbra. Although the cylinder and rotarod tests showed a trend of amelioration of behavioral impairments from the treatment, these were not statistically significant. Morphological brain injuries were not altered by treatment. The cell administration did not cause any adverse effects apart from hyperactivity in the open-field test. Some of these findings are consistent with the results obtained in our previous study using a stroke model, but others are not. This study suggests that the treatment protocol needs to be optimized for each pathological condition.

Silent information regulator 2 homolog 1 counters cerebral hypoperfusion injury by deacetylating endothelial nitric oxide synthase.

BACKGROUND AND PURPOSE: Silent information regulator 2 homolog 1 (SIRT1) is a protein deacetylase that has been reported to suppress neurodegenerative and cardiovascular diseases in model organisms. We hypothesized that neurovascular protection is one of the diverse actions of SIRT1. This study was designed to determine whether SIRT1 protects against the consequences of cerebral hypoperfusion in vivo. METHODS: Sirt1-overexpressing (Sirt1-Tg) mice driven by a prion promoter and their wild-type littermates were subjected to bilateral common carotid artery stenosis using external microcoils. Using Sirt1-Tg mice, we assessed the effect of SIRT1 on cerebral blood flow, cerebral angioarchitecture, histological and ultrastructural changes, and spatial working memory at several time points. We also evaluated the effects of preadministration of SIRT1 inhibitors or endothelial nitric oxide synthase inhibitors on cerebral blood flow after bilateral common carotid artery stenosis in Sirt1-Tg mice. Levels of acetylated and nonacetylated endothelial nitric oxide synthase were measured semiquantitatively with immunoblotting. RESULTS: Cerebral hypoperfusion induced by bilateral common carotid artery stenosis caused memory impairment and histological changes in wild-type littermates. However, these phenotypes were rescued in Sirt1-Tg mice, where cerebral blood flow was maintained even poststenosis. Electron microscopic analyses showed irregularities in the vascular endothelia, such as tight junction openings in wild-type mice, which were absent in Sirt1-Tg littermates. Brain endothelial nitric oxide synthase was acetylated after cerebral hypoperfusion in wild-type littermates but remained unacetylated in Sirt1-Tg mice. Moreover, treatment with SIRT1 inhibitors and endothelial nitric oxide synthase inhibitors abolished the vasculoprotective effects of SIRT1. CONCLUSIONS: Our results indicate that neurovascular endothelial SIRT1 potentiation upregulates the nitric oxide system and counters cerebral hypoperfusion injury. This novel cerebral blood flow-preserving mechanism offers potential molecular targets for future therapeutic intervention.

Hepatic arterial infusion of autologous CD34+ cells for hepatitis C virus-related decompensated cirrhosis: A multicenter, open-label, exploratory randomized controlled trial.

Introduction: In this multicenter clinical study, we aimed to investigate the efficacy and safety of the transhepatic arterial administration of granulocyte-colony stimulating factor (G-CSF)-mobilized autologous peripheral blood (PB)-CD34+ cells compared with standard therapy in patients with decompensated cirrhosis type C. Methods: Patients were randomly assigned (2:1) to the CD34+ cell transplant (CD34+ cell) or standard-of-care (SOC) group and followed up for 52 weeks. The primary endpoints were the non-progression rate of Child-Pugh (CP) scores at 24 weeks post-enrollment and the safety of the protocol treatment. Results: Fourteen patients (CD34+ cell group: 10; SOC group: 4) were enrolled. CP scores at 24 weeks had a non-progression rate of 90% in the CD34+ cell group and 100% in the SOC group, with no significant difference between groups. Importantly, 4 out of 10 patients in the CD34+ cell group exhibited an improvement from decompensated to compensated cirrhosis, whereas all patients in the SOC group remained in decompensated cirrhosis. With regard to secondary endpoints, a trend toward increased serum albumin levels in the CD34+ cell group was noted. Serious adverse events (SAEs) occurred in three patients in the CD34+ cell group and in one patient in the SOC group. No causal relationship was observed between all SAEs and G-CSF, leukapheresis, or cell transplantation in the CD34+ cell group. No patients died and no hepatocellular carcinoma occurred within the study period. Conclusions: PB-CD34+ cell infusion therapy may have the potential to circumvent the decompensated stage of cirrhosis, thus avoiding the need for liver transplantation.

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet.

A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specific to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin- and GC-C-specific siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

An in vitro culture platform to study the extracellular matrix remodeling potential of human mesenchymal stem cells.

The ability of mesenchymal stem cells (MSCs) to synthesize and degrade extracellular matrix (ECM) is important for MSC-based therapies. However, the therapeutic effects associated with ECM remodeling in cultured MSCs have been limited by the lack of a method to assess the ability of cultured cells to degrade ECM in vitro. Here, we describe a simple in vitro culture platform for studying the ECM remodeling potential of cultured MSCs using a high-density collagen (CL) surface. Cells on the CL surface have remarkable ability to degrade collagen fibrils by secreting matrix metalloproteinase (MMP); to study this, the marker collagen hybridizing peptide (CHP) was used. Confirming the ECM remodeling potential of MSCs with different population doublings (PDs), young and healthy γ-H2AX-negative cells, a marker of DNA damage and senescence, showed more extensive collagen degradation on the CL surface, whereas damaged cells of γ-H2AX-positive cells showed no collagen degradation. The frequency of γ-H2AX-/CHP + cells at PD = 0 was 49%, which was 4.9-fold higher than that at PD=13.07, whereas the frequency of γ-H2AX+/CHP- at PD=13.07 was 50%, which was 6.4-folds higher than that at PD=0. Further experimentation examining the in vitro priming effect of MSCs with the pro-inflammatory cytokine interferon-γ treatment showed increased frequency of cells with ECM remodeling potential with higher MMP secretion. Thus, this culture surface can be used for studying the ECM remodeling capacity of ex vivo-expanded MSCs in vitro and may serve as a platform for prediction in vivo ECM remodeling effect. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) remodeling potential of cultured mesenchymal stem cells (MSCs) is important for assessing the effectiveness of MSC-based therapy. However, methods to assess the ability of cultured cells to degrade ECM in vitro are still lacking. Here, we developed a simple in vitro culture platform to study the ECM remodeling potential of cultured MSCs using high-density collagen surfaces. This platform was used to evaluate the ECM remodeling potential of long-term ex vivo-expanded MSCs in vitro.

Transplantation of Human Brain-Derived Ischemia-Induced Multipotent Stem Cells Ameliorates Neurological Dysfunction in Mice After Stroke.

We recently demonstrated that injury/ischemia-induced multipotent stem cells (iSCs) develop within post-stroke human brains. Because iSCs are stem cells induced under pathological conditions, such as ischemic stroke, the use of human brain-derived iSCs (h-iSCs) may represent a novel therapy for stroke patients. We performed a preclinical study by transplanting h-iSCs transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion (MCAO). Compared with PBS-treated controls, h-iSC transplantation significantly improved neurological function. To identify the underlying mechanism, green fluorescent protein (GFP)-labeled h-iSCs were transplanted into post-stroke mouse brains. Immunohistochemistry revealed that GFP+ h-iSCs survived around the ischemic areas and some differentiated into mature neuronal cells. To determine the effect on endogenous neural stem/progenitor cells (NSPCs) by h-iSC transplantation, mCherry-labeled h-iSCs were administered to Nestin-GFP transgenic mice which were subjected to MCAO. As a result, many GFP+ NSPCs were observed around the injured sites compared with controls, indicating that mCherry+ h-iSCs activate GFP+ endogenous NSPCs. In support of these findings, coculture studies revealed that the presence of h-iSCs promotes the proliferation of endogenous NSPCs and increases neurogenesis. In addition, coculture experiments indicated neuronal network formation between h-iSC- and NSPC-derived neurons. These results suggest that h-iSCs exert positive effects on neural regeneration through not only neural replacement by grafted cells but also neurogenesis by activated endogenous NSPCs. Thus, h-iSCs have the potential to be a novel source of cell therapy for stroke patients.

Enhanced angiogenic properties of umbilical cord blood primed by OP9 stromal cells ameliorates neurological deficits in cerebral infarction mouse model.

Umbilical cord blood (UCB) transplantation shows proangiogenic effects and contributes to symptom amelioration in animal models of cerebral infarction. However, the effect of specific cell types within a heterogeneous UCB population are still controversial. OP9 is a stromal cell line used as feeder cells to promote the hematoendothelial differentiation of embryonic stem cells. Hence, we investigated the changes in angiogenic properties, underlying mechanisms, and impact on behavioral deficiencies caused by cerebral infarction in UCB co-cultured with OP9 for up to 24 h. In the network formation assay, only OP9 pre-conditioned UCB formed network structures. Single-cell RNA sequencing and flow cytometry analysis showed a prominent phenotypic shift toward M2 in the monocytic fraction of OP9 pre-conditioned UCB. Further, OP9 pre-conditioned UCB transplantation in mice models of cerebral infarction facilitated angiogenesis in the peri-infarct lesions and ameliorated the associated symptoms. In this study, we developed a strong, fast, and feasible method to augment the M2, tissue-protecting, pro-angiogenic features of UCB using OP9. The ameliorative effect of OP9-pre-conditioned UCB in vivo could be partly due to promotion of innate angiogenesis in peri-infarct lesions.

Systemic transplantation of allogenic fetal membrane-derived mesenchymal stem cells suppresses Th1 and Th17 T cell responses in experimental autoimmune myocarditis.

We have reported that systemic administration of autologous bone marrow or allogenic fetal membrane (FM)-derived mesenchymal stem cells (MSCs) similarly attenuated myocardial injury in rats with experimental autoimmune myocarditis (EAM). Since rat EAM is a T-helper (Th) cell-mediated autoimmune disease, and recent evidence has indicated that both autologous and allogenic MSCs exert an immunosuppressive effect on Th cell activity, we focused on Th cell differentiation in allogenic FM-MSC administered EAM rats. EAM was induced in Lewis rats by injecting porcine cardiac myosin (day 0). Allogenic FM-MSCs, obtained from major histocompatibility complex mismatched ACI rats, were intravenously injected (5 × 10(5)cells/rat) on days 7, 10, or 14 (MSCd7, MSCd10, or MSCd14 groups, respectively). At day 21, echocardiography confirmed that reduced ejection fraction in the untreated EAM group (63 ± 2%) was significantly improved in the MSCd10 and MSCd14 groups (74 ± 1 and 75 ± 2%, respectively, P<0.01). CD68 immunostaining revealed that prominent macrophage infiltration in the myocardium of the EAM group (1466 ± 93 cells/mm(2)) was significantly decreased in the MSCd10 group (958 ± 139 cells/mm(2), P<0.05). To evaluate Th cell differentiation, we used flow cytometry to determine the percentage of interferon (IFN)-γ positive Th1 and interleukin (IL)-17 positive Th17 cells in peripheral CD4-positive Th cells. The percentage of Th1 cells at day 16 was significantly lower in the MSCd10 (1.3 ± 0.2%) and MSCd14 (1.6 ± 0.3%) groups compared to the EAM group (2.4 ± 0.3%, P<0.05), as was the percentage of Th17 cells in the MSCd10 group (1.9 ± 0.5%) compared to the EAM group (2.2 ± 0.9%, P<0.05). At day 21, infiltrating Th17 cells in myocardium were significantly decreased in the MSCd10 group (501 ± 132 cells/mm(2), P<0.05) compared to EAM (921 ± 109 cells/mm(2)). In addition, human CD4+ Th cells co-cultured with human FM-MSCs exhibited reduced Th1 and Th17 cell-differentiation and proliferation, with increased expression of immunosuppressive molecules including indoleamine 2,3-dioxygenase 2 and IL-6 in co-cultured FM-MSCs. These results suggest that intravenous administration of allogenic FM-MSCs ameliorates EAM via the suppression of Th1/Th17 immunity.

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populations. METHODS: Given these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD model. RESULTS: Our study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD. CONCLUSIONS: Our results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.

Gene and protein expression analysis of mesenchymal stem cells derived from rat adipose tissue and bone marrow.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent and reside in bone marrow (BM), adipose tissue and many other tissues. However, the molecular foundations underlying the differences in proliferation, differentiation potential and paracrine effects between adipose tissue-derived MSC (ASC) and BM-derived MSC (BM-MSC) are not well-known. Therefore, we investigated differences in the gene and secretory protein expressions of the 2 types of MSC. METHODS AND RESULTS: ASC and BM-MSC were obtained from subcutaneous adipose tissue and BM of adult Lewis rats. ASC proliferated as rapidly as BM-MSC, and had expanded 200-fold in approximately 2 weeks. On microarray analysis of 31,099 genes, 571 (1.8%) were more highly (>3-fold) expressed in ASC, and a number of these genes were associated with mitosis and immune response. On the other hand, 571 genes (1.8%) were more highly expressed in BM-MSC, and some of these genes were associated with organ development and morphogenesis. In secretory protein analysis, ASC secreted significantly larger amounts of growth factor and inflammatory cytokines, such as vascular endothelial growth factor, hepatocyte growth factor and interleukin 6, whereas BM-MSC secreted significantly larger amounts of stromal-derived factor-1α. CONCLUSIONS: There are significant differences between ASC and BM-MSC in the cytokine secretome, which may provide clues to the molecule mechanisms associated with tissue regeneration and alternative cell sources.

Intravenous Administration of Human Amniotic Mesenchymal Stem Cells in the Subacute Phase of Cerebral Infarction in a Mouse Model Ameliorates Neurological Disturbance by Suppressing Blood Brain Barrier Disruption and Apoptosis via Immunomodulation.

Neuro-inflammation plays a key role in the pathophysiology of brain infarction. Cell therapy offers a novel therapeutic option due to its effect on immunomodulatory effects. Amniotic stem cells, in particular, show promise owing to their low immunogenicity, tumorigenicity, and easy availability from amniotic membranes discarded following birth. We have successfully isolated and expanded human amniotic mesenchymal stem cells (hAMSCs). Herein, we evaluated the therapeutic effect of hAMSCs on neurological deficits after brain infarction as well as their immunomodulatory effects in a mouse model in order to understand their mechanisms of action. One day after permanent occlusion of the middle cerebral artery (MCAO), hAMSCs were intravenously administered. RT-qPCR for TNFα, iNOS, MMP2, and MMP9, immunofluorescence staining for iNOS and CD11b/c, and a TUNEL assay were performed 8 days following MCAO. An Evans Blue assay and behavioral tests were performed 2 days and several months following MCAO, respectively. The results suggest that the neurological deficits caused by cerebral infarction are improved in dose-dependent manner by the administration of hAMSCs. The mechanism appears to be through a reduction in disruption of the blood brain barrier and apoptosis in the peri-infarct region through the suppression of pro-inflammatory cytokines and the M2-to-M1 phenotype shift.

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats.

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10(5) cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.

Allogenic fetal membrane-derived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis.

Mesenchymal stem cells (MSC) have been reported to be an attractive therapeutic cell source for the treatment of renal diseases. Recently, we reported that transplantation of allogenic fetal membrane-derived MSC (FM-MSC), which are available noninvasively in large amounts, had a therapeutic effect on a hindlimb ischemia model (Ishikane S, Ohnishi S, Yamahara K, Sada M, Harada K, Mishima K, Iwasaki K, Fujiwara M, Kitamura S, Nagaya N, Ikeda T. Stem Cells 26: 2625-2633, 2008). Here, we investigated whether allogenic FM-MSC administration could ameliorate renal injury in experimental glomerulonephritis. Lewis rats with anti-Thy1 nephritis intravenously received FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (FM-MSC group) or a PBS (PBS group). Nephritic rats exhibited an increased urinary protein excretion in the PBS group, whereas the FM-MSC group rats had a significantly lower level of increase (P < 0.05 vs. PBS group). FM-MSC transplantation significantly reduced activated mesangial cell (MC) proliferation, glomerular monocyte/macrophage infiltration, mesangial matrix accumulation, as well as the glomerular expression of inflammatory or extracellular matrix-related genes including TNF-α, monocyte chemoattractant protein 1 (MCP-1), type I collagen, TGF-ß, type 1 plasminogen activator inhibitor (PAI-1) (P < 0.05 vs. PBS group). In vitro, FM-MSC-derived conditioned medium significantly attenuated the expression of TNF-α and MCP-1 in rat MC through a prostaglandin E(2)-dependent mechanism. These data suggest that transplanted FM-MSC contributed to the healing process in injured kidney tissue by producing paracrine factors. Our results indicate that allogenic FM-MSC transplantation is a potent therapeutic strategy for the treatment of acute glomerulonephritis.