Mariko Omae

Subcutaneous adipose tissue–derived stem cells facilitate colonic mucosal recovery from 2,4,6‐trinitrobenzene sulfonic acid (TNBS)–induced colitis in rats

Background: Adipose tissue–derived stem cells (ADSCs) can be easily obtained from subcutaneous adipose tissue, and ADSCs can be demonstrated to display multilineage developmental plasticity. In this study, using TNBS‐induced colitis rats, we show the feasibility of repairing injured intestinal mucosa with adipose tissue–derived stem cells. Methods: The subcutaneous adipose tissue of F344 rats was obtained and digested by collagenase. The digested tissue was cultured in DMEM containing 10% FBS for 1 month. ADSCs were confirmed to differentiate under appropriate conditions into various lineages of cells, including bone, neural cells, adipocytes, and epithelial cells. HGF, VEGF, TGF‐&bgr;, and adiponectin in the culture supernatants of ADSCs were determined by ELISA. ADSCs (107 cells) were injected into the submucosa of the colon to examine their capacity to repair intestinal mucosa injured by TNBS. Results: In the experimental colitis model, the injection of ADSCs facilitated colonic mucosal repair and reduced the infiltration of inflammatory cells. High levels of HGF, VEGF, and adiponectin were detected in the culture supernatants of ADSCs. Moreover, injected ADSCs distributed to several layers of the colon, and some of them differentiated into mesodermal lineage cells. Conclusions: ADSCs can accelerate the regeneration of injured regions in experimental colitis. HGF, VEGF, and adiponectin might be responsible for the regeneration of injured regions in the colon.

Prevention of graft-versus-host disease by intra-bone marrow injection of donor T cells.

We have recently found that intra‐bone marrow‐bone marrow transplantation (IBM‐BMT) can be used to prevent graft‐versus‐host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM‐BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM‐BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV‐DLI]). GvHD in recipients treated with allogeneic IBM‐BMT plus IBM‐DLI was far milder than in those treated with allogeneic IBM‐BMT plus IV‐DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+CD25+ and CD4+Foxp3+ cells was significantly higher in recipients treated with IBM‐BMT plus IBM‐DLI than in those treated with IBM‐BMT plus IV‐DLI. Donor‐derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM‐BMT plus IBM‐DLI, whereas Th1 cells were dominant in recipients treated with IBM‐BMT plus IV‐DLI. Furthermore, the production of transforming growth factor‐β and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM‐DLI. Thus, IBM‐BMT plus IBM‐DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD.

Induction of Senile Osteoporosis in Normal Mice by Intra‐Bone Marrow‐Bone Marrow Transplantation from Osteoporosis‐Prone Mice

A P6 substrain of the senescence accelerated mouse (SAMP6) spontaneously develops osteoporosis early in life. These mice show the clinical signs of osteoporosis, such as elevated levels of urinary deoxypyridinoline (Dpd), decreased bone mineral density (BMD), and a significant loss of trabecular and cortical bone thickness at 12 months of age. Here, we describe the transfer of osteoporosis to a normal strain by the injection of bone marrow cells from SAMP6 donors directly into the bone marrow cavity (intra‐bone marrow‐bone marrow transplantation [IBM‐BMT]). More than 1 month after IBM‐BMT, hematolymphoid cells were completely reconstituted by donor‐derived cells, and bone marrow stromal cells that could differentiate into osteocytes were also found to be of donor origin. In addition, the recipient C57BL/6 mouse showed the features of osteoporosis in the trabecular bone. Decreases in BMD and increases in urinary Dpd were also observed. When the message levels of cytokines (interleukin [IL]‐11, IL‐6, receptor activator of NF‐κB ligand [RANKL], osteoprotegerin, macrophage–colony‐stimulating factor, and insulin‐like growth factor‐1) were examined by reverse transcription‐polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR analysis, IL‐6 and IL‐11 were reduced to a level similar to that in SAMP6 mice, whereas that of RANKL was increased. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment are reconstituted as a result of IBM‐BMT, and suggest that the development of senile osteoporosis might be attributable to “stem cell disorders.”

Kimura Disease: Diagnosis and Prognostic Factors

OBJECTIVES: To establish a preoperative diagnostic system and examine prognostic factors for Kimura disease. STUDY DESIGN: Retrospective study. SETTING: Hospital records were reviewed for nine cases of Kimura disease treated in our department. Preoperative eosinophil counts for 74 cases with untreated malignancy in the parotid gland were also examined. RESULTS: Parotid swelling with inhomogeneities and subcutaneous invasion on magnetic resonance imaging and eosinophils > 10.5 percent in Asian patients clearly indicates Kimura disease. Eosinophils > 50 percent, serum IgE levels > 10,000 IU/mL, and multifocal lesions outside salivary glands are prognostic factors suggesting disease recurrence. CONCLUSIONS: A preoperative decision based on our diagnostic criteria and prognostic factors should lead to better therapeutic outcomes for Kimura disease, for which a definitive treatment policy has never been determined.

The Wistar Bonn Kobori rat, a unique animal model for autoimmune pancreatitis with extrapancreatic exocrinopathy

The male Wistar Bonn/Kobori (WBN/Kob) rat is known to be a unique animal model for chronic pancreatitis with widely distributed fibrosis and degeneration of parenchyma because of the infiltration of lymphocytes. In this report, we show that female (but not male) rats develop dacryoadenitis at 3 months of age, and that both male and female WBN/Kob rats develop sialoadenitis, thyroiditis, sclerotic cholangitis and tubulointerstitial nephritis over 18 months of age. The infiltration of CD8+ cells and the deposits of tissue‐specific IgG2b were observed in the injured pancreas and lachrymal glands. Furthermore, the number of regulatory T cells (defined as CD4+ Forkhead box P3+ cells) decreased in the periphery of both male and female WBN/Kob rats, suggesting that the onset of these diseases is attributable, at least, to the failure in the maintenance of peripheral immune tolerance. These features show clearly that WBN/Kob rats are a useful animal model for autoimmune pancreatitis and Sjøgren‐like syndrome or multi‐focal fibrosclerosis in humans. We also show that these autoimmune diseases can be prevented by a newly devised strategy of bone marrow transplantation (BMT) in which bone marrow cells are injected directly into the bone marrow cavity: intrabone marrow–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.

Maintenance of systemic immune functions prevents accelerated presbycusis.

There is no effective therapy for progressive hearing loss such as presbycusis, the causes of which remain poorly understood because of the difficulty of separating genetic and environmental contributions. In the present study, we show that the age-related dysfunctions of the systemic immune system in an animal model of accelerated presbycusis (SAMP1, senescence-accelerated mouse P1) can be corrected by allogeneic bone marrow transplantation (BMT). We also demonstrate that this presbycusis can be prevented; BMT protects the recipients from age-related hearing impairment and the degeneration of spiral ganglion cells (SGCs) as well as the dysfunctions of T lymphocytes, which have a close relation to immune senescence. No donor cells are infiltrated to the spiral ganglia, confirming that this experimental system using BMT is connected to the systemic immune system and does not contribute to transdifferentiation or fusion by donor hematopoietic stem cells (HSCs), or to the direct maintenance of ganglion cells by locally infiltrated donor immunocompetent cells. Therefore, another procedure which attempts to prevent the age-related dysfunctions of the recipient immune system is the inoculation of syngeneic splenocytes from young donors. These mice show no development of hearing loss, compared with the recipient mice with inoculation of saline or splenocytes from old donors. Our studies on the relationship between age-related systemic immune dysfunctions and neurodegeneration mechanisms open up new avenues of treatment for presbycusis, for which there is no effective therapy.

Long-term maintenance of donor-derived hematopoiesis by intra-bone marrow-bone marrow transplantation.

The long-term maintenance of hematopoietic stem cells (HSCs) is assessed by serial bone marrow transplantation (BMT), in which HSCs are injected intravenously. Recently, we have found that intra-bone marrow (IBM)-BMT can efficiently reconstitute the hematopoietic system with cells of donor origin, in contrast to conventional intravenous (i.v.) BMT. In the present study, we have compared the long-term maintenance of HSCs using multiple rounds of serial i.v.-BMT and IBM-BMT. The frequencies of donor-derived progenitor cells (Lin(-)/c-kit(+) cells) and more primitive progenitors (Lin(-)/c-kit(+)/CD34(+)/Sca-1(+) cells) were higher in the tertiary recipients by serial IBM-BMT than in those that had received bone marrow cells by serial i.v.-BMT. Furthermore, neither donor-derived progenitor cells nor mature hematolymphoid cells were detected in approximately 25% of the tertiary recipients after serial i.v.-BMT, indicating that progenitor cells can be efficiently maintained by IBM-BMT but not by i.v.-BMT. Finally, we confirmed that the recipients treated with the primary IBM-BMT (without carrying out serial BMT) showed a significantly higher survival rate than those treated with i.v.-BMT. These findings clearly show that IBM-BMT efficiently promotes the longterm maintenance of donor-derived hematopoiesis.

Activation of granulocytes by direct interaction with dendritic cells

Granulocytes from human peripheral blood were co‐cultured with conventional dendritic cells (cDC) or plasmacytoid DCs (pDC) to examine the effects of DCs on the activation or function of granulocytes. After co‐culture of granulocytes with DCs, expression of the activation markers of granulocytes (CD63 and CD64) was up‐regulated, and increased expression of CD50, the activation marker and ligand for CD209 (DC‐SIGN) was also observed. The interaction of granulocytes with DCs was visualized as the cluster where DCs, especially cDCs, were surrounded by granulocytes to form a ‘rosette’. After co‐culture of granulocytes with cDCs, the secretion of elastase from granulocytes was enhanced significantly when examined cytohistochemically and by enzyme‐linked immunosorbent assay. An increase in myeloperoxidase (another activation index of granulocytes) was also observed after co‐culture with DCs. These findings suggest the functional and phenotypical activation of granulocytes by interaction with DCs. Furthermore, we examined the involvement of adhesion molecules in the granulocyte–DC interaction, and found that CD209 participates to some extent in this interaction.