ADVANCED MEDIA TECHNOLOGY

Transcription

1 ADVANCED MEDIA TECHNOLOGY For Human ES and ips Cell Research

2 The right medium makes all the difference in ensuring successful ES and ips cell culture. Stemgent offers high-quality, chemically-defined, ready-to-use media and related reagents for use with pluripotent stem cells. We have sourced and developed proven technologies with leading stem cell scientists to ensure that Stemgent media are the best available to allow stem cell cultures to thrive. Stemgent s complete medium, NutriStem XF/FF Culture Medium, was developed by Dr. Michal Amit in the laboratory of Professor Joseph Itskovitz-Eldor at the Technion Stem Cell Center, in collaboration with Biological Industries.

3 NutriStem XF/FF Culture Medium TESTED AND PROVEN Xeno-free, feeder-free, chemically-defined conditions containing no animal components Low basic FGF (4 ng/ml), and TGFβ (<5 ng/ml) allows for better control of human ES and ips cell culture conditions Easy, one-step transition from feeder-dependent culture, no adaptation required Maintains pluripotency, normal morphology, karyotype, and differentiation potential of human ES and ips cells over long term culture Robust attachment and high cloning efficiency from single cells Ideal for human ips cell generation with demonstrated high reprogramming efficiency in a completely xeno-free system 1 Product List: NutriStem XF/FF Culture Medium Cat. No Cat. No XENO-FREE AND FEEDER-FREE Conventional culture systems for the maintenance and expansion of pluripotent stem cells utilize undefined components, such as serum or serum-replacers along with feeder layers of mouse embryonic fibroblasts (MEFs). The use of these methods for human embryonic stem cell culture can be tedious and time-consuming and introduces variability, as performance can vary from batch-to-batch. In addition, the use of animal products may preclude stem cells from being suitable for clinical applications. NutriStem XF/FF Culture Medium is a chemically defined culture medium that contains human serum albumin (HSA) and enables culturing of human ES and ips cells under xenofree and feeder-free conditions thus eliminating the risk of contamination from animal components, reducing lot-to-lot variability, and resulting in predictable cellular behavior. DEFINED MEDIUM, LOW LEVELS OF CYTOKINES With NutriStem XF/FF Culture Medium, human ES and ips cells can be maintained and expanded readily under more physiological conditions than conventional methods. NutriStem XF/FF Culture Medium contains no inhibitors, cytokines, or growth factors other than low amounts of basic fibroblast growth factor (bfgf) (4 ng/ml) and transforming growth factor beta (TGFβ) (<5 ng/ml). Other commercially available feeder-free media contain levels of bfgf that are far higher. The effects of bfgf on the long term maintenance of pluripotent stem cells are unknown, and such conditions may be unfavorable to downstream experiments. At higher concentrations, bfgf is known to drive differentiation of pluripotent stem cells towards the neuro-ectoderm pathway. NutriStem XF/FF Culture Medium is ideal for maintaining and expanding human stem cells in long term culture (at least 50 passages have been tested) while retaining a normal morphology (Figure 1), karyotype (Figure 2), pluripotency marker expression (Figure 3), and the ability to differentiate into cells of all three germ layers in vitro and in vivo (Figure 4). Cells cultured for multiple passages in NutriStem XF/FF Culture Medium maintain typical morphology and proliferate at a rate that is equivalent to or greater than other commercially available feeder-free formulations (Figure 5). Human ES cells attach with very high efficiency after being passaged in NutriStem, resulting in more rapid expansion due to decreased loss of cells. Robust attachment is ideal for high-throughput screening applications, long-term maintenance and differentiation studies. 1. Shigeki Sugii, Yasuyuki Kida, Teruhisa Kawamura, Jotaro Suzuki, Rita Vassena, Yun-Qiang Yin, Margaret K. Lutz, W. Travis Berggren, Juan Carlos Izpisúa Belmonte, and Ronald M. Evans. (2010) Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells. PNAS, 107(8):

4 A B Figure 1. Morphology of human H1 ES cells in NutriStem XF/FF Culture Medium Figure 2. Human ES cells in long-term culture retain a normal karyotype in NutriStem XF/FF Culture Medium H1 human ES cells show a normal diploid karyotype by standard G-band analysis after 14 passages in NutriStem XF/FF Culture Medium

5 Figure 3. Human ES cells express pluripotency markers in NutriStem XF/FF Culture Medium Immunostaining of H1 human ES cells after 6 passages in NutriStem XF/FF Culture Medium shows expression of pluripotency markers Oct4 (A) and SSEA-4 (C). A) Oct 4 B) DAPI C) SSEA-4 D) DAPI Flow cytometry performed after 6 passages for pluripotency markers Oct4 (left) and SSEA-4 (right) shows that expression of these markers by cells cultured in NutriStem XF/FF Culture Medium is comparable to mtesr Oct SSEA-4 Counts Counts Isotype control NutriStem XF/FF Culture Medium mtesr1 Figure 4. Successful teratoma formation after culture with NutriStem XF/FF Culture Medium H1 human ES cells cultured with NutriStem XF/FF Culture Medium for 14 passages were injected subcutaneously into SCID mice. Teratomas were harvested after 8 weeks and processed for histological analysis. Tissue types from all three germ layers could be identified in the sections. (A) ectoderm with neural rosette (arrow marked N), (B) endoderm with columnar epithelium (arrow marked (E), and (C) mesoderm with cartilage (arrow marked C). N E C A B C Figure 5. Proliferation Growth rate of human H1 ES cells cultured in NutriStem XF/FF Culture Medium versus competitor mtesr1. Relative Cell Number 1 Hours

6 hes Cell Cloning & Recovery Supplement The hes Cell Cloning & Recovery Supplement is a multi-use human ES cell supplement that significantly increases the success of sub-cloning from single cells, improves thawing and attachment efficiency after passaging, and is a valuable tool when working with human ES cells under stressful conditions. The supplement contains the novel small molecule Thiazovivin in a convenient 1000x concentrate and is designed to be used directly in human ES cell culture. Thiazovivin has been shown to enhance the survival of human ES 2 cells and significantly improve the induction of human ips cells 3. Improves survival of single human ES cells by more than 30-fold after thawing, dissociation, and replating Considerably enhances cell attachment after routine passaging Culture remains xeno-free when used in combination with NutriStem XF/FF Culture Medium Product List: hes Cell Cloning & Recovery Supplement Cat. No C E L L P L AT I N G D E N S I T Y ( C E L L S / W E L L ) 2,500 5,000 10,000 20,000 40,000 + SUPPLEMENT SUPPLEMENT Figure 6. Growth of human ES cells after trypsinization with and without hes Cell Cloning and Recovery Supplement H1 cells were typsinized into single cells and re-seeded in NutriStem XF/FF Culture Medium with and without hes Cell Cloning and Recovery Supplement. 2 Yue X., Xiuwen, Z., Heung, S.H., Wanguo, W., Ergeng, H., Hayek, A., Ding, S. (2010) Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. PNAS, 107(23), induction of pluripotent stem cells. PNAS, 107(8): Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, Lin X, Hahm HS, Hao E, Hayek A, Ding S. (2009) A chemical platform for improved induction of human ipscs. Nat Methods. (11), Epub 2009 Oct 18. CryoStem Freezing Medium CryoStem Freezing Medium is a ready-to-use, animal component-free formulation, designed for the cryopreservation of human ES and ips cells. CryoStem has been tested on a number of stem cell lines that demonstrate higher rates of cell adherence and pluripotency marker expression following cryopreservation. Product List: CryoStem Freezing Medium Cat. No Xeno-free High cell viability and recovery with typical pluripotency marker expression after thawing Tested on human ES cells and mesenchymal stem cells

7 CellMates Accumax and Accutase Dissociation and Detachment Solutions CellMates Accumax Cell Dissociation and Accutase Cell Detachment Solutions are mixtures of proteolytic and collagenolytic enzymes that perform exceptionally well in dissociating cell clumps for cell counting, cell passaging, cell sorting, and flow cytometry, as well as bioreactor scale-up. Contain no components of mammalian or bacterial origin Gentler on cell membranes than conventional trypsin solutions Tested on human embryonic stem cells Stemolecules for the maintenance of human ES and ips cells Small molecules are increasingly used to replace cytokines and growth factors to generate more homogenous ES and ips cells. Stemolecules are of the highest purity (>98%), structurally verified by NMR and mass spectrometry, and tested on stem cells for cytotoxicity. Stemolecule Thiazovivin I Cat No Thiazovivin is a Rho-associated kinase (ROCK) inhibitor that dramatically improves the survival of human ES cells upon trypsinization. Thiazovivin in combination with inhibitors of the TGFβ receptor and the MEK pathway improve reprogramming efficiency more than 200-fold. Stemolecule Y27632 I Cat No Y27632 is a Rho-associated kinase (ROCK) inhibitor that enhances survival and cloning efficiency of human ES cells. Stemolecule GSK429286A I Cat No GSK429286A is a cell-permeable, selective small molecule inhibitor. Stemolecule Pyrintegrin I Cat No Pyrintegrin enhances the survival of human ES cells. Pyrintegrin has no effect on ROCK activity nor does it stabilize E-cadherin in human ES cells after trypsinization as thiazovivin does. Since the mechanism of action of pyrintegrin is different than inhibitors of the ROCK pathway, it may act synergistically with these molecules to further enhance reprogramming. Stemolecule ROCK I inhibitor I Cat No ROCK inhibitors, including ROCK I Inhibitor, have been found to prevent apoptosis, as well as enhance the survival and cloning efficiency of dissociated human embryonic stem ES cells without affecting their self-renewal properties or pluripotency. Stemolecule ROCK II inhibitor I Cat No ROCK II Inhibitor is a specific inhibitor for type 2 Rho-associated, coiled-coil containing protein kinase. ROCK inhibitors have been found to prevent apoptosis, as well as enhance the survival and cloning efficiency of dissociated human embryonic stem ES cells without affecting their self-renewal properties or pluripotency. Stemolecule CHIR99021 I Cat No CHIR99021 is the most selective inhibitor of GSK3β; self-renewal of embryonic stem cells is enabled by inhibition of GSK3β along with the elimination of differentiation-inducing signaling from mitogen-activated protein kinases. Stemolecule PD I Cat No PD selectively binds and inhibits MEK which likely inhibits the phosphorylation and activation of MAPK/ERK.

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