Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the Pax5 transgene

We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34+ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34+ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34+ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19+ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the κ/λ chain on Pax5-transduced CD19+ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors

Timing of Imaging after D-Luciferin Injection Affects the Longitudinal Assessment of Tumor Growth Using In Vivo Bioluminescence Imaging

The peak signal or the signal at a predetermined, fixed time point after D-luciferin injection may be used for the quantitative analysis of in vivo bioluminescence imaging. We repeatedly performed sequential bioluminescence imaging after subcutaneous injection of D-luciferin in mice bearing subcutaneous tumors. The peak time in each measurement became shorter early after cell inoculation, presumably due to gradual establishment of intratumoral vasculature, and reached a plateau of about 10 min on day 10. Although the correlation between the signal at a fixed time point and the peak signal was high, the signal at 5 or 10 min normalized for the peak signal was lower for earlier days, which caused overestimation of tumor growth. The time course of the signals after D-luciferin injection may vary with time after cell inoculation, and this variation should be considered when determining the imaging protocol for quantitative bioluminescence tumor monitoring

The Use of Bone Marrow Stromal Cells (Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells) for Alveolar Bone Tissue Engineering: Basic Science to Clinical Translation

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. Human bone marrow stromal cells (BMSCs) are the most commonly used cell source for bone tissue engineering. Although it is known that cell culture and induction protocols significantly affect the in vivo bone forming ability of BMSCs, the responsible factors of clinical outcome are poorly understood. The results from recent studies using human BMSCs have shown that factors such as passage number and length of osteogenic induction significantly affect ectopic bone formation, although such differences hardly affected the alkaline phosphatase activity or gene expression of osteogenic markers. Application of basic fibroblast growth factor helped to maintain the in vivo osteogenic ability of BMSCs. Importantly, responsiveness of those factors should be tested under clinical circumstances to improve the bone tissue engineering further. In this review, clinical application of bone tissue engineering was reviewed with putative underlying mechanisms

Adult T-Cell Leukemia/Lymphoma-Related Ocular Manifestations: Analysis of the First Large-Scale Nationwide Survey

Adult T-cell leukemia/lymphoma (ATL) is a rare and aggressive T-cell malignancy with a high mortality rate, resulting in a lack of information among ophthalmologists. Here, we investigated the state of ophthalmic medical care for ATL and ATL-related ocular manifestations by conducting the first large-scale nationwide survey in Japan. A total of 115 facilities were surveyed, including all university hospitals in Japan that were members of the Japanese Ophthalmological Society and regional core facilities that were members of the Japanese Ocular Inflammation Society. The collected nationwide data on the state of medical care for ATL-related ocular manifestations and ATL-associated ocular findings were categorized, tallied, and analyzed. Of the 115 facilities, 69 (60%) responded. Overall, 28 facilities (43.0%) had experience in providing ophthalmic care to ATL patients. ATL-related ocular manifestations were most commonly diagnosed “based on blood tests and characteristic ophthalmic findings.” By analyzing the 48 reported cases of ATL-related ocular manifestations, common ATL-related ocular lesions were intraocular infiltration (22 cases, 45.8%) and opportunistic infections (19 cases, 39.6%). All cases of opportunistic infection were cytomegalovirus retinitis. Dry eye (3 cases, 6.3%), scleritis (2 cases, 4.2%), uveitis (1 case, 2.1%), and anemic retinopathy (1 case, 2.1%) were also seen. In conclusion, intraocular infiltration and cytomegalovirus retinitis are common among ATL patients, and ophthalmologists should keep these findings in mind in their practice

Oncogenic fusion gene CD74-NRG1 confers cancer stem cell-like properties in lung cancer through a IGF2 autocrine/paracrine circuit

The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucinous adenocarcinoma, a malignant form of lung cancer. However, the function of the CD74-NRG1 fusion gene in adenocarcinoma pathogenesis and the mechanisms by which it may impart protumorigenic characteristics to cancer stem cells (CSC) is still unclear. In this study, we found that the expression of the CD74-NRG1 fusion gene increased the population of lung cancer cells with CSC-like properties. CD74-NRG1 expression facilitated sphere formation not only of cancer cells, but also of nonmalignant lung epithelial cells. Using a limiting dilution assay in a xenograft model, we further show that the CD74-NRG1 fusion gene enhanced tumor initiation. Mechanistically, we found that CD74-NRG1 expression promoted the phosphorylation of ErbB2/3 and activated the PI3K/Akt/NF-κB signaling pathway. Furthermore, the expression of the secreted insulin-like growth factor 2 (IGF2) and phosphorylation of its receptor, IGF1R, were enhanced in an NF-κB-dependent manner in cells expressing CD74-NRG1. These findings suggest that CD74-NRG1-induced NF-κB activity promotes the IGF2 autocrine/paracrine circuit. Moreover, inhibition of ErbB2, PI3K, NF-κB, or IGF2 suppressed CD74-NRG1-induced tumor sphere formation. Therefore, our study provides a preclinical rationale for developing treatment approaches based on these identified pathways to suppress CSC properties that promote tumor progression and recurrence. © 2016 American Association for Cancer Research

Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201 +CD150 +CD48 -c-Kit +Sca-1 +Lin - population of precultured HSCs. Using the Prkdc scid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy

Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

There is a high death rate of lung cancer patients. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in some lung adenocarcinoma patients with EGFR mutations. However, a significant number of patients show primary and acquire resistance to EGFR-TKIs. Although the Akt kinase is commonly activated due to various resistance mechanisms, the key targets of Akt remain unclear. Here, we show that the Akt-β-catenin pathway may be a common resistance mechanism. We analyzed gene expression profiles of gefitinib-resistant PC9M2 cells that were derived from gefitinib-sensitive lung cancer PC9 cells and do not have known resistance mechanisms including EGFR mutation T790M. We found increased expression of Axin, a β-catenin target gene, increased phosphorylation of Akt and GSK3, accumulation of β-catenin in the cytoplasm/nucleus in PC9M2 cells. Both knockdown of β-catenin and treatment with a β-catenin inhibitor at least partially restored gefitinib sensitivity to PC9M2 cells. Lung adenocarcinoma tissues derived from gefitinib-resistant patients displayed a tendency to accumulate β-catenin in the cytoplasm. We provide a rationale for combination therapy that includes targeting of the Akt-β-catenin pathway to improve the efficacy of EGFR-TKIs