Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

自動培養装置によるヒトiPS細胞の長期間培養に成功 . 京都大学プレスリリース. 2015-11-27.Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells

Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner

ヒト多能性幹細胞の拡大培養法の簡便かつ低コスト化に成功 : 培養基質のコーティングを必要としない培養法を開発. 京都大学プレスリリース. 2017-01-31.We describe highly effective adhesion culture of human pluripotent stem cells (hPSCs) using laminin fragments without precoating. Culture substrates have been generally thought to exert a cell adhesion effect when they are precoated onto culture vessels. However, simple addition of laminin fragments to a cell suspension during passaging accelerated the adhesion of single dissociated hPSCs onto culture vessels that were not precoated with any culture substrate. Interestingly, similar to conventional precoating, the uncoated addition of laminin fragments supported robust adhesion of single hPSCs and maximum adhesion at a much lower concentration compared with precoating. Similar to precoating laminin fragments, hPSCs seeded with uncoated laminin fragments grew well without cell detachment and maintained pluripotency after continuous subculture. We tested other culture substrates, including full-length laminin and vitronectin, to support hPSC adhesion in the uncoated manner, but only laminin fragments had the potential for application in the uncoated manner. This cost-effective and time-efficient method may contribute to expansion of culture of hPSCs and accelerate the development of regenerative medicine using hPSCs

Targeted Disruption in the MouseHoxc-4Locus Results in Axial Skeleton Homeosis and Malformation of the Xiphoid Process

AbstractHoxc-4is a mouse homeobox gene located at the 3′ end of theHoxCcluster. Of theHoxCgenes,Hoxc-4is expressed in the most anterior regions of the central nervous system and prevertebral column. To investigate its role in mouse development, we have generatedHoxc-4mutant mice by gene targeting. Mice homozygous for theHoxc-4mutation are viable and fertile. Analysis of the skeletal system of homozygous mutants revealed various abnormalities in the cervical and thoracic regions. The most frequent abnormality was a partial posterior homeotic transformation of the seventh cervical vertebra. Less frequently, anterior transformations of the third and eighth thoracic vertebrae were observed. Furthermore, the xiphoid process of the sternum was malformed such that it had an aperture or a fissure. AlthoughHoxc-4is expressed abundantly in the central nervous system, no obvious defects were observed. These results suggest thatHoxc-4is required for specifying cervical and thoracic vertebral identity

Overexpression of Nuclear Receptor 5A1 Induces and Maintains an Intermediate State of Conversion between Primed and Naive Pluripotency

Naive and primed human pluripotent stem cells (hPSCs) have provided useful insights into the regulation of pluripotency. However, the molecular mechanisms regulating naive conversion remain elusive. Here, we report intermediate naive conversion induced by overexpressing nuclear receptor 5A1 (NR5A1) in hPSCs. The cells displayed some naive features, such as clonogenicity, glycogen synthase kinase 3β, and mitogen-activated protein kinase (MAPK) independence, expression of naive-associated genes, and two activated X chromosomes, but lacked others, such as KLF17 expression, transforming growth factor β independence, and imprinted gene demethylation. Notably, NR5A1 negated MAPK activation by fibroblast growth factor 2, leading to cell-autonomous self-renewal independent of MAPK inhibition. These phenotypes may be associated with naive conversion, and were regulated by a DPPA2/4-dependent pathway that activates the selective expression of naive-associated genes. This study increases our understanding of the mechanisms regulating the conversion from primed to naive pluripotency

Efficient derivation and banking of clinical-grade human embryonic stem cell lines in accordance with Japanese regulations

[Introduction] We recently established clinical-grade human embryonic stem cell (hESC) line KthES11 in accordance with current good manufacturing practice standards in Japan. Despite this success, the establishment efficiency was very low at 7.1% in the first period. [Methods] To establish clinical-grade hESC lines, we used xeno-free chemically defined medium StemFit AK03N with the LM-E8 fragments instead of feeder cells. The protocol was then optimized, especially in the early culture phase. [Results] We established five hESC lines (KthES12, KthES13, KthES14, KthES15, and KthES16) with 45.5% efficiency. All five hESC lines showed typical hESC-like morphology, a normal karyotype, pluripotent state, and differentiation potential for all three germ layers. Furthermore, we developed efficient procedures to prepare master cell stocks for clinical-grade hESC lines and an efficient strategy for quality control testing. [Conclusions] Our master cell stocks of hESC lines may contribute to therapeutic applications using human pluripotent stem cells in Japan and other countries

Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells

Miyazaki, T. et al.. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat. Commun. 3:1236 doi: 10.1038/ncomms2231 (2012)

Distinct and Essential Roles of Transcription Factors IRF-3 and IRF-7 in Response to Viruses for IFN-α/β Gene Induction

AbstractInduction of the interferon (IFN)-α/β gene transcription in virus-infected cells is an event central to innate immunity. Mice lacking the transcription factor IRF-3 are more vulnerable to virus infection. In embryonic fibroblasts, virus-induced IFN-α/β gene expression levels are reduced and the spectrum of the IFN-α mRNA subspecies altered. Furthermore, cells additionally defective in IRF-7 expression totally fail to induce these genes in response to infections by any of the virus types tested. In these cells, a normal profile of IFN-α/β mRNA induction can be achieved by coexpressing both IRF-3 and IRF-7. These results demonstrate the essential and distinct roles of the two factors, which together ensure the transcriptional efficiency and diversity of IFN-α/β genes for the antiviral response

A Novel Serum-Free Monolayer Culture for Orderly Hematopoietic Differentiation of Human Pluripotent Cells via Mesodermal Progenitors

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation