Establishment and evaluation of the suspension culture system for umbilical cord- derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) derived from various tissues including bone marrow, adipose and umbilical cord tissues have been shown to modulate aberrantly activated immune system. With the features, MSC-based therapies targeting graft-versus-host disease (GvHD) by the administration of bone marrow-derived MSCs (BM-MSCs) have been available in some countries including Japan, and the expectations for the stable and cost effective supply system are getting higher and higher recently. However, the conventional culture systems which usually use plastic flask or multi-chamber equipment require space and manpower, thus the maximal expansion of MSCs at one production is likely to be limited. To compensate the limitation, repetitive productions have been unavoidable, and higher the production cost. Here, we introduced a new suspension-culture system, using micro-carriers and single-use-bioreactors, for the preparation of MSCs in anticipation of establishment of mass production system. Since the umbilical cord (UC) tissues can be collected through noninvasive procedure, and UC-derived MSCs (UC-MSCs) are shown to present higher proliferation rate and lower immunogenicity in comparison with BM-MSCs, we evaluated the potential and the versatility of UC-MSCs for the treatment of several diseases including GvHD. Results from several in vitro assays demonstrated that our new culture system maintains major key characteristics of MSCs, such as adhesiveness to cell culture surface, the expression of cell surface markers, differentiation capacities toward osteoblasts, chondroblasts, and adipocytes, and immunosuppressive effects on activated T cells. We are currently investigating cellular profiles and characteristics which are specific to the cells prepared in our suspension-culture system through meta-analysis. The established suspension-culture system is presumed to attain the mass production of UC-MSCs, contributing to lower the cost and also providing possible applications for MSCs from other origins

Characterization of the chromosomal inversion associated with the Koa mutation in the mouse revealed the cause of skeletal abnormalities

<p>Abstract</p> <p>Background</p> <p>Koala (<it>Koa</it>) is a dominant mutation in mice causing bushy muzzle and pinna, and is associated with a chromosomal inversion on the distal half of chromosome 15. To identify the gene responsible for the <it>Koa </it>phenotypes, we investigated phenotypes of <it>Koa </it>homozygous mice and determined the breakpoints of the inversion with a genetic method using recombination between two different chromosomal inversions.</p> <p>Results</p> <p>Skeletal preparation of <it>Koa </it>homozygotes showed marked deformity of the ribs and a wider skull with extended zygomatic arches, in addition to a general reduction in the lengths of long bones. They also had open eyelids at birth caused by a defect in the extension of eyelid anlagen during the embryonic stages. The proximal and distal breakpoints of the <it>Koa </it>inversion were determined to be 0.8-Mb distal to the <it>Trsps1 </it>gene and to 0.1-Mb distal to the <it>Hoxc4 </it>gene, respectively, as previously reported. The phenotypes of mice with the recombinant inverted chromosomes revealed the localization of the gene responsible the <it>Koa </it>phenotype in the vicinity of the proximal recombinant breakpoint. Expression of the <it>Trsps1 </it>gene in this region was significantly reduced in the <it>Koa </it>homozygous and heterozygous embryos.</p> <p>Conclusion</p> <p>While no gene was disrupted by the chromosomal inversion, an association between the <it>Koa </it>phenotype and the proximal recombinant breakpoint, phenotypic similarities with <it>Trps1</it>-deficient mice or human patients with <it>TRSP1 </it>mutations, and the reduced expression of the <it>Trsps1 </it>gene in <it>Koa </it>mice, indicated that the phenotypes of the <it>Koa </it>mice are caused by the altered expression of the <it>Trps1 </it>gene.</p

c-Src activation as a potential marker of chemical-induced skin irritation using tissue-engineered skin equivalents.

Skin irritancy to topically applied chemicals is a significant problem that affects millions of people worldwide. New or modified chemical entities must be tested for potential skin irritancy by industry as part of the safety and toxicity profiling process. Many of these tests have now moved to a non-animal-based format to reduce experiments on animals. However, these tests for irritancy potential often rely on monolayer cultures of keratinocytes that are not representative of the skin architecture or tissue-engineered human skin equivalents (HSE) using complex multi-gene expression panels that are often cumbersome and not amenable for high throughput. Here, we show that human skin equivalents increase abundance of several phosphorylated kinases (c-Src, c-Jun, p53, GSK3α/β) in response to irritant chemical stimulation by phosphokinase array analysis. Specific phosphorylation of c-SrcY419 was confirmed by immunoblotting and was plasma membrane-associated in basal/spinous cells by phospho-specific immunohistochemistry. Moreover, c-SrcY419 phosphorylation in response to the irritants lactic acid and capsaicin was inhibited by the c-Src inhibitors KB-SRC and betaine trimethylglycine. These data provide the first evidence for c-Src specific activation in response to chemical irritants and point to the development of new modes of rapid testing by immunodetection for first-pass screening of potential irritants