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International Society for Stem Cell Research (ISSCR) Annual Meeting
Do-it-yourself, ready-made, and custom products & services for stem cell research
The International Society for Stem Cell Research (ISSCR) is the premier organization that connects scientists from around the world to share their research and expertise and advance the fields of stem cell research and regenerative medicine. Adding to the body of expertise at ISSCR, Takara Bio offers attendees guidance, technologies, and services for advancing their stem cell studies. We have a broad portfolio of ready-made and do-it-yourself products for stem cell research. Additionally, we offer custom services for sourcing, banking, reprogramming, differentiation, and clinical-grade hES cell line derivation so you can focus on your areas of expertise while we take care of the rest.
We're looking forward to seeing you at ISSCR 2020. In the meantime, we invite you to review the materials we presented at past ISSCR meetings and reach out to us with any questions or requests via the "speak with us" link below.
ISSCR 2019: posters
Robust differentiation procedure into human induced pluripotent stem cell-derived endothelial cells and their properties
Primary human endothelial cells (ECs) such as HUVECs are widely used for vascularization study, various organoids formation or safety/toxicological test. However, primary ECs sometimes show uncontrollable lot-to-lot variances in these researches due to genetic diversity of derived donors, and thereby may lead to the difficulty of obtaining the reproducibility of the experiment. Human induced pluripotent stem cell-derived ECs (iPS-ECs) are expected to use for these research field as cell source having stable properties among manufacturing batches from single donor. Nevertheless, iPS-ECs have not been widely utilized in those experiments, since current iPS-ECs have some problems such as unstable differentiation from iPS cells or poor growth capacity. To overcome this, we have developed robust differentiation procedure into iPS-ECs in which continuous proliferation has been observed. We tested their ability of the procedure to differentiate into ECs from various iPS cell clones. All iPS-ECs from tested iPS cell clones showed more than 90% of both CD31 and CD144 positive population, which are typical endothelial cell surface markers. Then, these iPS-ECs were evaluated for their growth capacity for one month. Although all iPS-ECs exhibited almost similar endothelial cell-related genes and markers expression, there were big differences in the proliferation rate. The most proliferated iPS-ECs showed about one thousand fold expansion, even though one out of 6 iPS-ECs could little proliferate after differentiation (about a few fold expansion). In spite of the differences in growth capacity, all iPS-ECs retained both CD31 and CD144 positive population during observation period. We also confirmed whether iPS-ECs could be used for angiogenesis inhibition assay by observing the tube formation by measuring the fluorescence of calcein-labeled ECs. As a result, the inhibition of tube formation was observed in the addition of Wortmannin in a dose-dependent manner. Overall we have successfully developed iPS-ECs differentiation system. It can be overcome the lot variation problems of primary endothelial cells, making them suitable for various kinds of vascularization research.
Establishing human embryonic stem cell master cell banks under GMP conditions
The increased interest in using human pluripotent stem cells for advanced therapy medicinal products (ATMP) has revealed the urgent need for safe and GMP compliant established hESC Master Cell Banks. The culture conditions for human pluripotent stem cells have evolved since 1998, leaving the use of mouse feeders and animal components behind, and instead one employ defined, feeder free culture conditions. However, the absolute majority of the derived human ES cells are made with feeders and outside the GMP environment, with animal components. Further, the starting material, the blastocysts, are not sourced according to FDA's guidelines, lacking proper donor testing, and/or sourced in non-prion free regions of the world. In order to derive human embryonic stem cells under GMP and sourcing the starting material according to also FDA's requirement we have built up the infrastructure required for a successful establishment of human ES cells for clinical applications. Initially we developed a new, xeno-free defined culture medium, that together with a defined coating substrate was successfully evaluated for derivation of hES cells. The xeno-free medium was then produced and released under GMP. We then established appropriate sourcing procedures, e.g., sourcing according to FDA's requirement and with proper donor consents that allows the future use and needed analyses as well as significant prescreening of the donor couples before they were approved as eligible donors. In parallel we built the infrastructure and processes that rendered us to become a registered tissue establishment and with a manufacturing license for deriving and banking human pluripotent stem cells from the Swedish MPA (according to the Swedish LVFS 20018:12 and LVFS 2004:7, Eudralex Volume 4 GMP). We will present the workflow as well as data from the ongoing derivation work.
ISSCR 2018: posters
Newly optimized medium for the expansion of human pluripotent stem cell-derived endothelial cells
Human endothelial cells (ECs) are widely used in the field of vascularization studies such as drug discovery, safety/toxicological test, tumor growth or tissue engineering including organ-on-a-chip technology. Although primary ECs like HUVECs are generally used for these purposes, there are some drawbacks in utilizing primary cells because of the donor difference due to a wide variety of genetic background. To overcome this, human pluripotent stem cell-derived ECs (hPS-ECs) are expected to be supplied as stable cell source with similar properties and without the donor difference among manufacturing batches. We have already developed the differentiation method from pluripotent stem cells into ECs population having more than 95% of CD31 and CD144 positive cells, which are identified as endothelial cells. Our hPS-ECs also express CD34 in more than 95% of cells, suggesting immature phenotype. This is considered to be one of the appropriate features for tissue engineering study because ECs may be needed to be educated to fit in each tissue. However, it was difficult to obtain a large number of hPS-ECs by using commercially available medium for primary ECs due to poor proliferation capacity, even though many cells are required for tissue engineering. Thus, we have also developed the culture medium for hPS-ECs. As a result, it was observed that hPS-ECs show about 10-fold expansion in one week by newly optimized medium. The expanded hPS-ECs maintained ECs surface marker (CD31 and CD144) expression similar to the cells in pre-expansion. Furthermore, tube-like structure formation on matrigel was also observed after expansion, indicating an angiogenic capacity of hPS-ECs. Overall we successfully developed hPS-ECs culture system using newly optimized medium. The cells and medium can overcome the lot variation problems of primary endothelial cells, making them suitable for industrialization with mass cell production. Further, we are currently developing xeno-free medium for hPS-ECs to apply to the manufacture of regenerative medicine.
Newly developed xeno-free medium for human mesenchymal stem cells show robust cell-expansion capacity
Human mesenchymal stem cells (hMSCs) are an attractive candidate for cell therapy due to their multipotential differentiation activities into each cell type or immunomodulatory properties. For therapeutic applications, hMSCs are needed to be expanded to appropriate cell number because primary hMSCs obtained from bone marrow, adipose or cord blood are usually limited and thereby the required cell number can't be obtained. Although many medium for culturing hMSCs are currently proposed, further improvements in their cell-expansion capacity or the maintenance of multipotential differentiation activities have been needed. Furthermore, medium for clinical use should be at least xeno-free formulation because of the potential risks of FBS, such as virus or prion contamination. Thus, we have newly developed xeno-free medium for hMSCs, having especially robust cell-expansion capacity. Both bone-marrow and adipose-derived hMSCs expansions using new medium were several-fold higher than those using conventional other medium (commercially available). Moreover, new medium could be used even in the extracellular matrix coating-free condition. After several passages, we confirmed the expression of cell surface markers identified as hMSCs such as CD73, CD90, and CD105 by flow cytometry and colony-forming unit-fibroblast (CFU-F) capacity of the expanded cells. The cells expanded by new medium were shown to be almost 100% of CD73, CD90, and CD105 positive population, but a little decrease of CD105 expression was observed in the cells by other medium. Since CFU-F capacity was almost equal among tested medium, the obtained colony-forming cells in new medium were several-fold larger than those in other medium. We have also confirmed their multipotential differentiation activities into adipocytes, chondrocytes, and osteocytes. Overall, it is considered that our newly developed xeno-free medium has the desired properties to expand hMSCs for therapeutic application.
Expansion system for producing a large amount (>10^9 cells) and high purity (>90%) of human CD3-CD56+NK cells from PBMCs and their therapeutic application due to ADCC activity in xenogenic mouse model
Human natural killer (NK) cells are one of the attractive candidates for cell-based therapy against any cancers due to their strong cytotoxicity. However, there are few convenient and efficient method to obtain a large amount and high purity of functional NK cells from peripheral blood mononuclear cells (PBMCs) derived from a small amount of blood. Thus, we have developed a robust NK-cell expansion method using OK-432, IL-2 and RetroNectin induced T (RN-T) cells as a stimulator. RN-T cells were prepared by previously established co-stimulation method using anti-CD3mAb and RetroNectin and treated to suppress the growth potential (modified RN-T cells). NK cells could be expanded from PBMCs stimulated with modified RN-T cells, OK-432 and IL-2, then cultured for more than 16 days. In our large-scale culture system using gas-permeable culture bag (CultiLife 215 and CultiLife Eva), we could obtain 109–1010 cells containing a high proportion (>90%) of CD3-CD56+ NK cells from 50 ml of peripheral blood. Furthermore, almost all cells displayed functional cell surface molecules such as NKG2D and CD16 implicated in cytotoxicity and antigen-dependent cell cytotoxicity (ADCC). Thus, we investigated the antitumor effect of the expanded NK cells combined with Trastuzumab against HER2-positive human gastric cancer cell line NCI-N87 in hIL-2 Tg NOG mice (hIL-2-NOG mice; Central Institute for Experimental Animals). In this experiment, we used purified NK cells to reduce GVHD risk caused by human CD3+ cells including in the expanded cells. As a result, the combination of the NK cells and Trastuzumab dramatically enhanced the antitumor activity compared with each treatment alone. The chimerism of human NK cells in mouse peripheral blood was observed during the observation period without any GVHD symptoms, and functional NK-cell surface markers such as CD16 and NKG2D also expressed in human NK cells. Furthermore, human NK cells were observed into tumor tissue even 3 months after administration. Overall, we have established a robust NK-cell expansion system, and the expanded cells showed strong antitumor activity in a xenogenic mouse model. It is considered that our expansion system could be used for chimeric antigen receptor (CAR)-NK cell processing or pluripotent stem cell-derived NK-cell manufacture for future application.
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