Extracellular Vesicles and Cx43-Gap Junction Channels Are the Main Routes for Mitochondrial Transfer from Ultra-Purified Mesenchymal Stem Cells, RECs.

Mitochondria are essential organelles for maintaining intracellular homeostasis. Their dysfunction can directly or indirectly affect cell functioning and is linked to multiple diseases. Donation of exogenous mitochondria is potentially a viable therapeutic strategy. For this, selecting appropriate donors of exogenous mitochondria is critical. We previously demonstrated that ultra-purified bone marrow-derived mesenchymal stem cells (RECs) have better stem cell properties and homogeneity than conventionally cultured bone marrow-derived mesenchymal stem cells. Here, we explored the effect of contact and noncontact systems on three possible mitochondrial transfer mechanisms involving tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels (GJCs), and extracellular vesicles (Evs). We show that Evs and Cx43-GJCs provide the main mechanism for mitochondrial transfer from RECs. Through these two critical mitochondrial transfer pathways, RECs could transfer a greater number of mitochondria into mitochondria-deficient (ρ0) cells and could significantly restore mitochondrial functional parameters. Furthermore, we analyzed the effect of exosomes (EXO) on the rate of mitochondrial transfer from RECs and recovery of mitochondrial function. REC-derived EXO appeared to promote mitochondrial transfer and slightly improve the recovery of mtDNA content and oxidative phosphorylation in ρ0 cells. Thus, ultrapure, homogenous, and safe stem cell RECs could provide a potential therapeutic tool for diseases associated with mitochondrial dysfunction.

Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer.

BACKGROUND: Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer. METHODS: We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors. RESULTS: Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore. CONCLUSIONS: RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.

Simultaneous Discordant B-Lymphoblastic Lymphoma and Follicular Lymphoma.

OBJECTIVES: We report a rare case of B-lymphoblastic lymphoma (B-LBL) and low-grade follicular lymphoma (FL) identified concurrently in biopsies from different sites at the initial diagnosis in a 39-year-old man. The clonal relationship between the 2 histologic subtypes was investigated. METHODS: A diagnosis of FL grade 1/2 (low grade) was made by bone marrow (BM) biopsy. B-LBL was identified in biopsies from the testis and pancreas. Cytogenetic and molecular analyses were performed to investigate their clonal relationship. RESULTS: Interphase fluorescence in situ hybridization analyses and G-banding karyotype analyses identified the BCL2-IGH and MYC-IGH translocation in tumor cells from both the BM and testis. The tumor cells from the BM and testis shared the same IGH VDJ usage and a high degree of somatic mutations. These findings suggest that acquisition of MYC gene rearrangement is a critical event for lymphoblastic transformation of FL. Of note, the presence of intraclonal diversity in the B-LBL sample further suggests an earlier or concurrent event of MYC translocation than the somatic IGH mutation in the germinal center and the dedifferentiation of lymphoma cells to a precursor stage of B-cell development. CONCLUSIONS: B-lymphoblastic transformation of FL can occur with MYC gene rearrangement.

Dose-adjusted EPOCH with or without rituximab for aggressive lymphoma patients: real world data.

CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) -/+ rituximab (R) is the standard chemotherapeutic regimen for aggressive lymphoma, but is insufficient for aggressive lymphoma with adverse prognostic factors. Dose-adjusted (DA)-EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisolone) -/+ R demonstrates excellent efficacy against some aggressive lymphoma. Thus, we conducted a retrospective study to evaluate the feasibility and efficacy of this therapy in clinical practice. We enrolled 149 patients from 17 institutions diagnosed between 2007 and 2015. The median follow-up period for survivors was 27 months (range 0.2-123). The complete response (CR) rate of newly diagnosed patients was 79% (95% CI 68-87%). All patients were hospitalized to receive this therapy and 94% of patients also received granulocyte-colony-stimulating factor support. There were no treatment-related deaths. Febrile neutropenia (FN) and grade 3 or 4 infection occurred in 55% and 28% of patients, respectively. There were no significant differences in FN or infection between young (≤ 65 years) and elderly patients (> 65 years). In newly diagnosed diffuse large B-cell lymphoma-not otherwise specified patients (n = 46), the CR rate was 80% (95% CI 64-91%) and the 2-year OS rate was 81% (95% CI 66-90%). In the present study, DA-EPOCH -/+ R exhibited excellent efficacy and feasibility for aggressive lymphoma.

[Cooperation between Clinics and Hospitals Using a Critical Path for G-CSF Prophylaxis in Cancer Chemotherapy].

BACKGROUND: Prophylactic granulocyte-colony stimulating factor(G-CSF)is necessary for some cancer patients receiving anti-cancer drugs. However, it is difficult for cancer patients in rural areas to receive G-CSF as outpatients because of inconvenient official transport, lack of public support, and low activity levels due to age. To resolve this problem, we began conducting a critical path(G-path)with regional medical institutions from 2011. METHODS: We retrospectively surveyed the clinical records of cancer patients receiving prophylactic G-CSF using G-path at our hospital. RESULTS: Eighty-two patients who were administered a total of 254 cycles of chemotherapy were examined between January 2011 and December 2016. Diseases included malignant lymphoma(n=64), pancreatic cancer(n=7), soft tissue sarcoma(n=5), and others(n=6). The median age of the patients was 70(range: 24-94)years. Fifty-three patients visited medical offices, and 31 patients visited regional hospitals. In 245 of 254(96%)cycles, planned G-CSF administration was performed. In 37 of 254(15%)cycles, infectious episodes developed, but patients needed hospitalization for only 5 cycles(2%). CONCLUSION: Cooperation between clinics and hospitals using G-path reduced ambulatory burden and prevented severe infection. Cooperation in supportive care may allow for equal accessibility to cancer treatment.