Immune Dysfunction Associated with Abnormal Bone Marrow-Derived Mesenchymal Stroma Cells in Senescence Accelerated Mice.

Senescence accelerated mice (SAM) are a group of mice that show aging-related diseases, and SAM prone 10 (SAMP10) show spontaneous brain atrophy and defects in learning and memory. Our previous report showed that the thymus and the percentage of T lymphocytes are abnormal in the SAMP10, but it was unclear whether the bone marrow-derived mesenchymal stroma cells (BMMSCs) were abnormal, and whether they played an important role in regenerative medicine. We thus compared BMMSCs from SAMP10 and their control, SAM-resistant (SAMR1), in terms of cell cycle, oxidative stress, and the expression of PI3K and mitogen-activated protein kinase (MAPK). Our cell cycle analysis showed that cell cycle arrest occurred in the G0/G1 phase in the SAMP10. We also found increased reactive oxygen stress and decreased PI3K and MAPK on the BMMSCs. These results suggested the BMMSCs were abnormal in SAMP10, and that this might be related to the immune system dysfunction in these mice.

Stem Cell Replacement Improves Expression of SMP30 in db/db Mice.

We have previously reported that replacing bone marrow stem cells may improve hyperglycemia and oxidative stress in db/db mice, a type 2 diabetic mouse model. Senescence marker protein 30 (SMP30) is an antioxidant protein that decreases with aging. However, it has not been clear whether SMP30 decreases in the livers of obese mice, and whether stem cell replacement would improve SMP30 expression in the liver. Bone marrow stem cells of db/db mice were replaced with the bone marrow stem cells of C57BL/6 mice. Plasma cytokine and insulin levels were measured, and glycogen content, expression of SMP30, and fibrosis in the liver were assessed. Our results showed that stem cell replacement increased the expression of SMP30 in the liver, resulting from decreased plasma inflammation cytokines and hyperinsulinemia in db/db mice. This is the first report that stem cell replacement increased the expression of SMP30 in the liver, and may help prevent fibrosis in the liver of db/db mice.

Stem cell treatment for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and neurodegenerative disorder that induces dementia in older people. It was first reported in 1907 by Alois Alzheimer, who characterized the disease as causing memory loss and cognitive impairment. Pathologic characteristics of AD are ß-amyloid plaques, neurofibrillary tangles and neurodegeneration. Current therapies only target the relief of symptoms using various drugs, and do not cure the disease. Recently, stem cell therapy has been shown to be a potential approach to various diseases, including neurodegenerative disorders, and in this review, we focus on stem cell therapies for AD.

[Efficacy of mitral valve repair for anterior leaflet prolapse of mitral valve].

OBJECTIVE: To evaluate the therapeutic effects of mitral valve repair for the treatment of the anterior leaflet prolapse of mitral valve. METHODS: From November 1998 to October 2007, 210 patients with severe anterior leaflet prolapse of mitral valve underwent valve repair. The condition of valve was preoperative, intraoperative, and postoperative assessed with echocardiography. RESULTS: Edge-to-edge repair technique was used in 134 cases (63.8%). The cardiac function was NYHA class I in 168 cases and class II in 40 cases after operation. Patients were followup for 1 - 150 (25.7 +/- 29.0) months, two patients (0.95%) died of postoperative low cardiac output syndrome. Echocardiography examination indicated that the mean JP2 postoperative left atrial diameter was (37.7 +/- 9.2) mm against the preoperative value of (47.5 +/- 12.7) mm (P < 0.05), the mean postoperative left ventricular end-diastolic diameter was (51.7 +/- 7.9) mm against the preoperative value of (67.7 +/- 10.3) mm (P < 0.05), the mean postoperative left ventricular ejection fraction was (62.2 +/- 3.2)% against the preoperative value of (52.2 +/- 6.4)% (P < 0.05), and the mean preoperative regurgitation area was (10.4 +/- 4.1) cm(2) against the postoperative value of (4.1 +/- 1.7) cm(2) (P < 0.01). CONCLUSIONS: Optimal outcome was achieved by appropriate edge-to-edge technique or other mitral valve repair techniques for anterior leaflet prolapse of mitral valve. Edge-to-edge technique is a reliable and efficient surgical technique.

Analysis of tolerance induction using triple chimeric mice: major histocompatibility complex-disparate thymus, hemopoietic cells, and microenvironment.

BACKGROUND: Although bone marrow transplantation (BMT) has become a valuable strategy for the treatment of various intractable diseases in recent years, success rates remain low in elderly patients because of low thymic function. We have previously shown that fetal thymus transplantation (TT) with BMT is effective for elderly recipients in mice. METHODS: We performed fully major histocompatibility complex (MHC)-mismatched fetal TT from B6 (H-2) mice plus allogeneic BMT from C3H/HeN (H-2) mice by intra-bone marrow-BMT (IBM-BMT) using congenitally athymic nude (nu/nu) BALB/c (H-2), or BALB/c adult-thymectomized recipients to obtain triple chimeras. We next carried out the IBM-BMT+TT using senescence-accelerated mouse P1 strain (SAMP1) to examine whether this method would be applicable to aging mice. RESULTS: Triple chimeric mice survived for a long period with sufficient T-cell functions comparable to the mice treated with BMT plus MHC-matched TT, whereas those without TT survived for a short period with insufficient T-cell reconstitution. Almost all the hematolymphoid cells were derived from donor bone marrow cells. Interestingly, they showed tolerance to all three types of MHC determinants with donor-derived thymic dendritic cells in TT. Triple chimeric SAMP1 also survived for long periods with T-cell functions restored in contrast to non-TT SAMP1 recipients. CONCLUSION: These findings suggest that third party combined TT with allogeneic IBM-BMT may be more advantageous for elderly recipients with low thymic function, than IBM-BMT alone (without TT).

Long-term donor-specific tolerance in rat cardiac allografts by intrabone marrow injection of donor bone marrow cells.

BACKGROUND: Donor-specific central tolerance in cardiac allograft can be induced by hematopoietic chimerism via conventional intravenous bone marrow transplantation (IV-BMT). However, there are problems with IV-BMT, such as the risk of graft failure and of the toxicity from conditioning regimens. METHODS: A new method for heart transplantation is presented. This method consists of administration of fludarabine phosphate (50 mg/kg) and fractionated low-dose irradiation (3.5 Gyx2 or 4.0 Gyx2), followed by intrabone marrow injection of whole bone marrow cells (IBM-BMT) plus heterotopic heart transplantation. RESULTS: Cardiac allografts with IBM-BMT were accepted and survived long-term (>10 months) showing neither acute rejection nor chronic rejection including cardiac allograft vasculopathy by such conditioning regimens. In contrast, cardiac allografts with conventional IV-BMT were rejected within 1 month after the treatment with irradiation of 3.5 Gyx2 or within 3 months after the treatment with irradiation of 4.0 Gyx2. Macrochimerism (>70%) was favorably established and stably maintained by IBM-BMT but not IV-BMT. Low levels of transient mixed chimerism (<7%) were induced by IV-BMT with fludarabine plus 4.0 Gyx2, but the chimerism was lost within 1 month after the treatment. CONCLUSIONS: These findings indicate that IBM-BMT is a feasible strategy for the induction of persistent donor-specific tolerance, enables the use of reduced radiation doses as conditioning regimens, and obviates the need for immunosuppressants.

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow-bone marrow transplantation.

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow-bone marrow transplantation: IBM-BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2-3 days old) donor mice is also effective in the IBM-BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM-BMT+bone grafts, but not in the mice that had received IBM-BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM-BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.

Interdiction of the diabetic state in NOD mice by sustained induction of heme oxygenase: possible role of carbon monoxide and bilirubin.

The aims of the present study were to assess whether sustained HO-1 expression could moderate or prevent diabetes in an animal model of the disease and, if so, to examine the possible mechanisms involved. Our results showed that HO-1 expression and HO activity were upregulated in the pancreas of non-obese diabetic (NOD) mice by the weekly administration of cobalt protoporphyrin (CoPP). Blood glucose levels in CoPPtreated mice decreased to normal, but continuously increased in untreated controls. Beta-cell numbers were preserved in the islets of CoPP-treated mice, whereas no beta cells were found in untreated diabetic mice. The number of CD11c(+) dendritic cells was significantly decreased in the pancreas of CoPP-treated NOD mice, but this effect was reversed by the inhibition of HO activity. Increased levels of HO-1 produced a new pancreatic phenotype, as reflected by increases in phosphorylated AKT, BcL-xL and RSK levels, and decreases in O(2)- and 3-NT levels. These novel findings provide a link between the increase in HO-1 activity, with its concurrent enhanced production of carbon monoxide (CO) and bilirubin, a decrease in infiltrated CD11c(+) dendritic cells and an increase in anti-apoptotic proteins, including RSK and BcL-xL, in the interdiction of the diabetic state.

Prevention of osteoporosis and hypogonadism by allogeneic ovarian transplantation in conjunction with intra-bone marrow-bone marrow transplantation.

BACKGROUND: We investigated the effects of ovarian allograft in conjunction with intra-bone marrow-bone marrow transplantation (IBM-BMT) on estrogen deficiency in mice. METHODS: Female C57BL/6 mice underwent ovariectomy (OvX). After 3 months, the mice were irradiated at 9.5 Gy, and the bone marrow cells (BMCs) of female BALB/c mice (8 weeks old) were then injected into the bone cavity of the B6 mice. Simultaneously, allogeneic ovaries from BALB/c mice were transplanted under the renal capsules of the B6 mice. RESULTS: Three months after the transplantation, the hematolymphoid cells were found to be completely reconstituted with donor-derived cells. The transplanted ovary tissues under the renal capsules were accepted without using immunosuppressants; there were a large number of growing follicles at different stages of development. Atrophic endometrium and its glands were also recovered by ovarian transplantation (OT). The transplanted allogeneic ovaries secreted estrogen at normal levels. Furthermore, bone loss was prevented to a certain extent. CONCLUSIONS: These findings suggest that IBM-BMT+OT will become a valuable strategy for young women with malignant tumors to prevent premature senescence, including hypogonadism and osteoporosis, after radiochemotherapy.

Stem cell transplantation upregulates Sirt1 and antioxidant expression, ameliorating fatty liver in type 2 diabetic mice.

Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance, oxidative stress, and obesity. The db/db mouse model displays increased levels of insulin resistance, obesity, and an over-accumulation of hepatic triglycerides, making it an excellent model for studying NAFLD. In db/db mice, intra-bone marrow-bone marrow transplantation plus thymus transplantation (IBM-BMT+TT) improves type 2 diabetes mellitus (T2 DM) by normalizing the T-cell imbalance. We hypothesized that this approach would improve Sirt1 expression in the liver and benefit liver development. The db/db mice were treated with IBM-BMT+TT, and plasma MCP-1, IL-6, adiponection, LDL, Sirt1, and HO-1 levels were then assessed. Stem cell transplantation decreased the levels of plasma inflammatory cytokines and LDL while it increased the expression of Sirt1 and HO-1, resulting in decreased progression of fatty liver. Moreover, Sirt1 and HO-1 expression were both detected in the thymus and many HO-1-positive cells were observed in the bone marrow. This is the first report of stem cell transplantation improving the antioxidant function in the liver, thymus, and bone marrow of db/db mice by increasing the levels of Sirt1 and HO-1. This approach may prove useful in the treatment of nonalcoholic steatohepatitis and its clinical manifestations.

Mesenchymal stem cells for inducing tolerance in organ transplantation.

Organ transplantation is useful for treating the end stage of organ failure. The induction of tolerance to the transplanted organ is essential for its long-term survival. Immunologic tolerance can be induced by immunosuppressive agents and mixed chimerism. Mixed chimerism is a state in which both recipient-and donor-derived blood cells remain in the hematopoietic system after allogeneic hematopoietic stem cells have been transplanted. Mesenchymal stem cells (MSCs), and immune cells such as dendritic cells and T-reg cells play an important role in the induction of tolerance. MSCs secrete cytokines, which modulate the immune response. In particular, they upregulate T-reg cell function and thereby induce tolerance. Intra-bone marrow-bone marrow transplantation recruits both donor-derived HSCs and MSCs, inducing persistent donor-specific tolerance without the use of immunosuppressants. In this review, we summarize the use of MSCs to induce tolerance in organ transplantation.

Long-term results of a minimally invasive surgical pulmonary vein isolation and ganglionic plexi ablation for atrial fibrillation.

BACKGROUND: Ganglionated plexi (GP) ablation has been become an adjunct to pulmonary vein isolation (PVI). This study describes the long-term results of minimally invasive surgical PVI, ablation of GPs, and exclusion of the left atrial appendage for atrial fibrillation (AF). METHODS: Long-term follow-up of 55 months was performed in 139 consecutive patients (age 58.3±20.8 years) with symptomatic, drug-refractory lone AF who underwent minimally invasive surgical PVI, GPs ablation, and exclusion of the left atrial appendage. Success was defined as freedom from AF, atrial flutter, or atrial tachycardia off antiarrhythmic drugs. RESULTS: AF was paroxysmal in 77.7%, persistent in 12.2% and long-standing persistent in 10.1%. Single-procedure success rate was 71.7%, 59.4% and 46.6% at 12, 24 and 60 months respectively. Single-procedure success rate was 72.9%, 62.6% and 51.8% for paroxysmal AF, 64.7%, 35.3%, and 28.2% for persistent AF, 71.4%, 64.3% and 28.6% for long-standing persistent AF at 12, 24 and 60 months respectively. Duration of AF>24 months (hazard ratio [HR]: 3.09, 95% confidence interval [CI]: 1.51 to 6.32; p = 0.002), left atrial diameter≥40 mm (HR: 4.03, 95% CI: 1.88 to 8.65; p<0.001), early recurrence of AF (HR: 4.66, 95% CI: 2.25 to 9.63; p<0.001) independently predicted long-term recurrence of AF. There was no procedure-related death. One patient converted to median sternotomy because of uncontrolled bleeding. Two patients underwent perioperative cerebrovascular events. CONCLUSIONS: At nearly 5-year of clinical follow-up, single-procedure success rate of minimally invasive surgical PVI with GP ablation was 51.8% for paroxysmal AF, 28.2% for persistent AF, 28.6% for long-standing persistent AF after initial procedure. Patients with AF duration≤24 months, left atrial diameter<40 mm and no early recurrence of AF, had favorable outcomes.

A successful haploidentical bone marrow transplantation method in rabbits: perfusion method plus intra-bone marrow-bone marrow transplantation.

Graft versus host disease (GVHD), rejection, delayed immune reconstitution and infections have been significant hurdles to haploidentical BMT. In order to improve the outcome of the current haploidentical-related BMT, we performed a novel BMT method consisting of the perfusion method (PM) plus intra-bone marrow-bone marrow transplantation (IBM-BMT) in a rabbit model. The percentages of T cells in BMCs harvested by the PM and the conventional aspiration method (AM) were 6% and 14%, respectively (p<0.01). Conversely, the CFU-C counts of BMCs in the PM group were significantly higher than those in the AM group. When the BMCs were transplanted into lethally irradiated offspring rabbits by IBM-BMT, hemopoietic recovery in the PM group was faster than in the AM group. The cumulative incidence of acute GVHD was 25% in the PM group versus 75% in the AM group (p<0.05). In addition, the survival rate was 75% in the PM group versus 33% in the AM group (p<0.05). Thus, the new method is able to provide rapid hemopoiesis, reduce the cumulative incidence of acute GVHD, and achieve a higher survival rate. This novel strategy paves the way for new dimensions in haploidentical BMT.

Prevention of premature ovarian failure and osteoporosis induced by irradiation using allogeneic ovarian/bone marrow transplantation.

BACKGROUND: Two side effects of irradiation are premature ovarian failure (POF) and osteoporosis, both of which are concerns not only clinically, for patients, but also experimentally, for animals. We examine whether bone marrow transplantation (BMT) can correct the POF induced by radiation and also address whether allogeneic ovarian transplantation (OT) can modulate the adverse effects of radiotherapy. METHODS: Eight-week-old female C57BL/6 mice were lethally irradiated with 6 Gy x2, and then injected with allogeneic bone marrow cells into their bone marrow cavity using our previously described intrabone marrow (IBM)-BMT technique. Allogeneic ovaries were simultaneously transplanted under the renal capsules of the mice. RESULTS: Three months after the transplantation, we noted that hematopoietic and lymphoid cells had been successfully reconstituted. The ovaries transplanted under the renal capsules demonstrated signs of development with a large number of differentiating follicles at different stages of development. Importantly, the total bone mineral density of the tibia in the "IBM-BMT+OT" (BMT/OT) group remained normal. However, the reproductive function of the recipient mice was not restored, despite the presence of many immature oocytes in the host ovaries in the BMT/OT group. In the BMT group, no oocytes were found in the host ovaries. CONCLUSIONS: These findings suggest that IBM-BMT with ovarian allografts can be advantageous for young women with POF and osteopenia or osteoporosis that is due to chemotherapy and radiotherapy for malignant diseases.

Amelioration of cognitive ability in senescence-accelerated mouse prone 8 (SAMP8) by intra-bone marrow-bone marrow transplantation.

Bone marrow cells (BMCs) can increase the number of activated microglias, which play a central role in the inflammatory response in Alzheimer's disease (AD). Senescence-accelerated mouse (SAM) prone 8 (SAMP8) are widely used in various experiments because of cognitive deficits observed with age. In the present study, 4-month-old SAMP8 were reconstituted with BMCs of C57BL/6 mice by intra-bone marrow-bone marrow transplantation (IBM-BMT), which can reconstitute both donor-derived hemopoietic stem cells and mesenchymal stem cells. Three months after IBM-BMT, the impairment of spatial memory in SAMP8 was found to be ameliorated after analyzing the results of the water maze test. Although IL-1beta, IL-6 and iNOS increased and TGF-beta decreased in 7M SAMP8, IL-1beta, IL-6 and iNOS decreased while TGF-beta increased after IBM-BMT by RT-PCR. Moreover, oxidative stress-related heme oxygenase-1 (HO-1) increased in 7M SAMP8, but significantly decreased after IBM-BMT. In conclusion, this is the first report suggesting that the impaired cognitive ability of SAMP8 is ameliorated by IBM-BMT. It seems likely that decreases in IL-1beta, IL-6, iNOS and HO-1 are a result of the development of donor-derived BMCs.

Long-lasting expression of HO-1 delays progression of type I diabetes in NOD mice.

Heme oxygenase-1 (HO-1) is crucial in regulating oxidative injury. The present study was designed to assess whether HO-1 upregulation by cobalt protoporphyrin IX (CoPP) moderates or prevents the diabetic state in non-obese diabetic (NOD) mice, an animal model for Type 1 diabetes (T1D). HO-1 expression and HO activity were upregulated in the pancreas by the intermittent administration of CoPP. This was associated with decreases in blood glucose and pancreatic O2-, but increased pAKT and BcL-XL and cell survival. A considerable number of beta cells were preserved in the islets of CoPP-treated NOD mice, while none were found in untreated diabetic mice. The number of CD11c+ dendritic cells was decreased in the pancreas of CoPP-treated NOD mice (p < 0.05). These novel findings provide a link between the increase in HO-1 and a decrease in infiltrated CD11c+ dendritic cells, and suggest that induction of HO-1 activity can be used to enhance cell survival and moderate the diabetic state in T1D.

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes.

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra-bone-marrow (IBM) route (IBM-BMT) over the intravenous route (IV-BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT or IV-BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor-derived cells in total and of c-kit(+) cells were observed at 2 through 6 days after IBM-BMT. In parallel, significantly higher numbers of colony-forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM-BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM-BMT over IV-BMT. Disclosure of potential conflicts of interest is found at the end of this article.