Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4–7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-β-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts.

Background: Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma.

Background and Purpose— Data on the prevalence of Fabry disease in patients with central nervous system pathology are limited and controversial. In this study, we assessed the prevalence of Fabry disease in young patients presenting with cerebrovascular disease in Belgium. Methods— In this national, prospective, multicenter study, we screened for Fabry disease in 1000 patients presenting with ischemic stroke, transient ischemic attack, or intracranial hemorrhage; unexplained white matter lesions; or vertebrobasilar dolichoectasia. In male patients, we measured α-galactosidase A (α-GAL A) activity in dried blood spots. Female patients were screened for mutations by exonic DNA sequencing of the α-GAL A gene. Results— α-GAL A activity was deficient in 19 men (3.5%), although all had normal α-GAL A gene sequences. Enzymatic deficiency was confirmed on repeat assessment in 2 male patients (0.4%). We identified missense mutations in 8 unrelated female patients (1.8%): Asp313Tyr (n=5), Ala143Thr (n=2), and Ser126Gly (n=1). The pathogenicity of the 2 former missense mutations is controversial. Ser126Gly is a novel mutation that can be linked to late-onset Fabry disease. Conclusion— α-GAL A deficiency may play a role in up to 1% of young patients presenting with cerebrovascular disease. These findings suggest that atypical variants of Fabry disease with late-onset cerebrovascular disease exist, although the clinical relevance is unclear in all cases.

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Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.

Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells.

Background: Pretreated advanced melanoma is a poor prognosis scenario with few, if any, active therapeutic options. The antibody against vascular endothelial growth factor, bevacizumab, has demonstrated increased activity in combination with chemotherapy in many tumors. We intended to evaluate the activity of the combination of weekly paclitaxel and bevacizumab in previously treated metastatic melanoma. Patients and Methods: Patients with previously treated metastatic melanoma received paclitaxel 70 mg/m² weekly and bevacizumab 10 mg/kg biweekly for 5 consecutive weeks every 6 weeks. Results: Twelve patients were treated. Two patients (16.6%) achieved a partial response and 7 patients (58.3%) stable disease. Responses were seen in soft tissue, lung and brain metastases. Median disease-free and overall survival times were 3.7 and 7.8 months, respectively. Treatment was well tolerated. Main toxicities were grade 3 asymptomatic lymphopenia in 6 patients, grade 3 leucopenia in 2 patients, and grade 3 thrombocytopenia in 1 patient. Conclusions: Our preliminary results suggest that the combination of bevacizumab and weekly paclitaxel is active and safe in patients with metastatic melanoma, warranting further investigation.

Recent studies suggest that signal transduction may have an important role in the development and regulation of the metastatic phenotype. Here, we investigated the role of the epidermal growth factor receptor (EGFR), and protein kinase C (PKC), in the process of reassembly of cadherin-dependent cell-cell adhesion of Caco-2 cells. We used chemical activation of PKC and EGFR with 12- O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent, pretreatment with protein kinase inhibitors and subcellular fractionation to analyze the effect of the phorbol ester on the redistribution of junctional proteins. Transepithelial resistance (TER), electron microscopy and immunofluorescence analyses were also carried out. Activation with TPA resulted in disassembly of adherens junctions (AJs), but the tight junction (TJ) structure and function remained unaltered. TPA affected E-cadherin levels. In Caco-2 cells at day 2 of culture, when most E-cadherin is not associated with the cytoskeleton, a decrease in the level of this protein was observed as soon as 6 h after TPA addition. However, at day 5 of culture, the major effect observed after 6 h of treatment was a translocation of the protein from the Triton-insoluble to the -soluble fraction. On the other hand, TPA did not significantly affect the E-cadherin-associated proteins alpha and beta-catenins. Potent specific EGFR inhibitors, such as PD153035 and Tyrphostin 25, as well as Calphostin C, an inhibitor of PKC, significantly blocked the effect of TPA on AJs. Furthermore, inhibition of the TPA effect by the PD98059 MAPK inhibitor suggests that activation of this kinase was the final event in the modulation of cadherin-dependent cell-cell adhesion. Pretreatment of cell monolayers with Calphostin C before EGF treatment, one of the ligands of EGFR, blocked the redistribution of E-cadherin caused by EGF. Based on these results, we conclude that both EGFR and PKC activation are involved in TPA-induced cell signaling for modulation of cadherin-dependent cell-cell adhesion and cell shape in Caco-2 cells.