Kequan Guo

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

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.

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 Gy×2 or 4.0 Gy×2), 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 Gy×2 or within 3 months after the treatment with irradiation of 4.0 Gy×2. 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 Gy×2, 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.

Extensive Studies on Perfusion Method Plus Intra‐Bone Marrow‐Bone Marrow Transplantation Using Cynomolgus Monkeys

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra‐bone marrow (IBM) injection of BMCs (termed IBM‐bone marrow transplantation) has also been confirmed using 30 monkeys.

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 β-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.

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.

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 Alzheimers 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.

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-2b) mice plus allogeneic BMT from C3H/HeN (H-2k) mice by intra-bone marrow-BMT (IBM-BMT) using congenitally athymic nude (nu/nu) BALB/c (H-2d), 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).

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.

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 ×2, 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.