Teruyuki Kajiume

Novel Electrical Stimulation Sets the Cultured Myoblast Contractile Function to ‘On’

Objective: In the present study, the effect of electrical stimulation was examined for the ability to induce morphological, physiological, and molecular biological effects on myoblasts during cell differentiation. Methods: L6 rat myoblasts were electrically stimulated by newly developed methods on culture days 6, 8, 10 and 12. Results: This electrical stimulation accelerated the appearance of myotubes, and subsequently produced spontaneously contracting muscle fibers. Measurement of membrane potential showed that the contracting cell had functional ion channels and gap junctional intercellular communication. In the electrically stimulated cells, an enhanced expression of MyoD family and M-cadherin was also observed. Expression of connexin 43 was increased and maintained at a high level in the electrically stimulated cells. Conclusion: This is the first demonstration of in vitro induction of myoblasts in spontaneously contractile muscle fibers by intermittent stimulation. This novel method for induction of myoblast differentiation represents an important advance in cell therapy.

LIF-free embryonic stem cell culture in simulated microgravity.

Background Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. Methodology/Principal Findings We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. Conclusions/Significance Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.

Allogeneic peripheral blood stem cell transplantation in children with hematologic malignancies from HLA-matched siblings

BACKGROUND Despite the ethical problem of using granulocyte colony-stimulating factor (G-CSF) in normal children, allogeneic peripheral blood stem cell transplantation (PBSCT) might have advantages over allogeneic bone marrow transplantation (BMT). PROCEDURE Eleven HLA-matched sibling donors aged 2-16 years received 10 microg/kg/day G-CSF for 5 days and underwent apheresis to harvest peripheral blood stem cells (PBSC). PBSC were then cryopreserved until infusion. The 11 corresponding patients aged 8 months to 14 years with high-risk hematological malignancies received busulfan (16 mg/kg or 600 mg/m(2)) and melphalan (210 mg/m(2)) as a preparative regimen. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methylprednisolone. RESULTS All of the donors tolerated G-CSF administration and apheresis procedures. The patients received a median of 5.8 (range 1. 4-11.5) x 10(6)/kg CD34(+) cells, 17.2 (3.8-36.0) x 10(5)/kg colony forming units-granulocyte/macrophage (CFU-GM), and 3.5 (1.4-7.1) x 10(8)/kg CD3(+) cells. All of the patients showed prompt engraftment, with a median time to reach an absolute neutrophil count (ANC) above 0.5 x 10(9)/liter of 10 (9-13) days. Grade I acute GVHD occurred in seven patients (64%), whereas grade II-IV acute GVHD was not seen. Chronic GVHD occurred in four patients (40%) among 10 patients evaluable for chronic GVHD. Three patients showed extensive chronic GVHD. Currently, eight patients (73%) are alive and disease-free for a median follow-up of 775 (103-1,069) days. CONCLUSIONS Allogeneic PBSCT is feasible in the pediatric population, and PBSC harvest is an alternative to BM harvest in donors who are not eligible for BM harvest. Furthermore, PBSC were successfully collected in pediatric donors with peripheral access. The choice of a stem cell source should be based on the risk/benefit assessment for both patients and donors.

Simulated microgravity maintains the undifferentiated state and enhances the neural repair potential of bone marrow stromal cells.

Recently, regenerative medicine with bone marrow stromal cells (BMSCs) has gained significant attention for the treatment of central nervous system diseases. Here, we investigated the activity of BMSCs under simulated microgravity conditions. Mouse BMSCs (mBMSCs) were isolated from C57BL/6 mice and harvested in 1G condition. Subjects were divided into 4 groups: cultured under simulated microgravity and 1G condition in growth medium and neural differentiation medium. After 7 days of culture, the mBMSCs were used for morphological analysis, reverse transcription (RT)-polymerase chain reaction, immunostaining analysis, and grafting. Neural-induced mBMSCs cultured under 1G conditions exhibited neural differentiation, whereas those cultured under simulated microgravity did not. Moreover, under simulated microgravity conditions, mBMSCs could be cultured in an undifferentiated state. Next, we intravenously injected cells into a mouse model of cerebral contusion. Graft mBMSCs cultured under simulated microgravity exhibited greater survival in the damaged region, and the motor function of the grafted mice improved significantly. mBMSCs cultured under simulated microgravity expressed CXCR4 on their cell membrane. Our study indicates that culturing cells under simulated microgravity enhances their survival rate by maintaining an undifferentiated state of cells, making this a potentially attractive method for culturing donor cells to be used in grafting.

Reciprocal expression of Bmi1 and Mel-18 is associated with functioning of primitive hematopoietic cells

OBJECTIVE The Polycomb-group (PcG) genes regulate global gene expression in many biological processes, including hematopoiesis, by manipulating specific target genes. It is known that various PcG genes regulate self-renewal of hematopoietic stem cells (HSCs). Here we have shown that the reciprocal expression of PcG proteins regulates self-renewal and differentiation of HSCs. METHODS We used murine and human bone marrow cells and evaluated the reciprocal expression of PcG proteins on the basis of their respective intranuclear distributions. PcG-gene expression in HSCs was knocked down by small interfering RNAs. The function of each gene in HSCs was analyzed in vitro and in vivo. RESULTS Cells were either Bmi1-positive or Mel-18-positive. The Bmi1-positive cells contained very little amounts of Mel-18 and vice versa. The bmi1-knockdown marrow cells did not show HSC function, while the mel-18-knockdown marrow cells showed increased stem cell function. Results of the analysis on human cells were similar to those observed in case of murine cells. In a clinical investigation, transplantation using sources with a low Bmi1 to Mel-18 ratio was associated with early hematopoietic recovery. CONCLUSION Reciprocal expression of Bmi1 and Mel-18 regulated HSC function. Here, we observed that expression of the PcG genes-bmi1 and mel-18-is correlated with self-renewal and differentiation of HSCs. Thus, it was suggested that the balance between Bmi1 and Mel-18 regulates self-renewal of HSCs.

Electrical stimulation enhances neurogenin2 expression through β-catenin signaling pathway of mouse bone marrow stromal cells and intensifies the effect of cell transplantation on brain injury.

Bone marrow stromal cells (BMSCs) have received significant attention for its use in neural regeneration. However, neural replacement by transplanted BMSCs was not very effective. Recently, the gene transfection method has improved the capability of cell transplantation; however, this method results in canceration and immune rejection. We induced the differentiation of mouse BMSCs into neural cells using electrical stimulation and transplanted the cells into traumatic brain injury (TBI) model mice. We found that the electrically stimulated cells have good potential to differentiate into neural cells and contribute to recovery from TBI without differentiating into astrocytes. In addition, we found that electrical stimulation enhanced neurogenin2 (Ngn2) expression. Ngn2 is involved in neural differentiation and inhibits astrocytic differentiation during cell growth. Furthermore, we found that this enhancement of Ngn2 expression occurred through β-catenin signaling pathway. This study may contribute to the use of BMSCs for neural replacement in central nervous system diseases.

Intra-apheresis recruitment of blood progenitor cells in children

BACKGROUND: Determination of the optimal duration of apheresis requires a careful examination of blood progenitor cell (BPC) kinetics during apheresis. Intra‐apheresis recruitment of BPCs should be evaluated.

Steroid-Dependent ACTH-Produced Thymic Carcinoid: Regulation of POMC Gene Expression by Cortisol via Methylation of Its Promoter Region

Aims: Metyrapone causes a decrease in the serum cortisol level without affecting ACTH production in ectopic tumors. We report a case who presented with Cushing’s syndrome due to an ectopic ACTH-producing thymic carcinoid. In the present case, it was demonstrated that metyrapone administration resulted in a significant decrease in the plasma ACTH and serum cortisol levels. We hypothesized that the steroid hormone may promote proopiomelanocortin (POMC) gene expression in the carcinoid cells. Methods: An 11-year-old boy presented with Cushing’s syndrome. Prior to the detection of a thymic tumor, metyrapone was administered to ameliorate the symptoms of Cushing’s syndrome. Interestingly, plasma ACTH as well as serum cortisol levels immediately decreased after metyrapone administration. The levels of cortisol and ACTH were observed to be normal after complete surgical resection of the tumor. Biological characterization of the tumor cells was by in vitro analysis. Results: Thein vitro culture of the tumor cells showed an increased expression of POMC in the presence of cortisol. A CpG methylation assay showed that the demethylation of the POMC promoter was induced by a steroid hormone. Conclusion: These findings suggest that the ectopic ACTH-producing tumor may partly be regulated by the elevated levels of cortisol.

A case of myelodysplastic syndrome complicated by pulmonary alveolar proteinosis with a high serum KL-6 level.

Serious hematological diseases often cause respiratory disorders. Because these are related to the prognoses of patients with hematological diseases, their early diagnosis is necessary. This study describes a 6-year-old girl with myelodysplastic syndrome complicated by pulmonary alveolar proteinosis who showed a remarkable increase in her serum KL-6 level. Three years and 2 months after the end of therapy for neonatal melanoma, a diagnosis of myelodysplastic syndrome with leukemic change was made. Ten months after the onset of leukemia, she had respiratory distress with an increased serum KL-6 level of 75,000 U/mL (reference range; < 500 U/mL). Despite various treatments for pulmonary complications, she died 3 months after developing respiratory distress. A diagnosis of pulmonary alveolar proteinosis was made at autopsy. Earlier treatment of respiratory distress could be achieved if serum KL-6 levels were examined earlier.

Continuous Intravenous Infusion of Ketamine and Lidocaine as Adjuvant Analgesics in a 5-Year-Old Patient with Neuropathic Cancer Pain

For difficult to treat neuropathic pain from cancer, adjuvant analgesics are often used with opioids. We present the case of a 5-year-old girl who was diagnosed with meningitis caused by malignant T-cell lymphoma. She had severe neuropathic pain not relieved by increasing doses of a fentanyl infusion. Intravenous administration of ketamine and lidocaine in combination with fentanyl provided excellent analgesia without significant side effects. Ketamine and lidocaine can be safely infused together with concomitant opioids for the treatment of refractory neuropathic pain caused by cancer.