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Definitions

• the present disclosure relates, in various exemplary embodiments, generally to compositions and methods for enabling adhesion, proliferation and self- renewal maintenance of pluripotent, or undifferentiated, human stem cells grown in vitro.
• human stem cells include embryonic stem cells, bone marrow stem cells, and pluripotent stem cells.
• a stem cell is an undifferentiated cell from which specialized cells are subsequently derived.
• Embryonic stem cells possess extensive self-renewal capacity and pluripotency with the potential to differentiate into cells of all three germ layers. They are useful for therapeutic purposes and may provide unlimited sources of cells for tissue replacement therapies, drug screening, functional genomics and proteomics. (Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
• feeder cells for maintenance in a pluripotent state in vitro or differentiation inhibitors like Noggin and/or high doses of basic fibroblast growth factor (FGF) when cultured on MatrigelTM (see for review: Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
• FGF basic fibroblast growth factor
• the use of feeder cells has a number of drawbacks.
• feeder cells can contain pathogens, such as viruses that can infect the stem cells (Hovatta, O., and Skottman, H.
• Feeder-free systems that support human embryonic stem cell self-renewal require either i) MatrigelTM (Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C, and Bongso, A. (2002); Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells; Nat Biotechnol 20, 933-936); (Xu, C, Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D., and Carpenter, M. K.
• Extracellular matrix (ECM) proteins particularly basement membrane (BM) components, constitute an important part of in vivo niches for differentiation, phenotype maintenance and function of many types of somatic and stem cells in the human body and, therefore, have a potential for self- renewal of certain cell types.
• basement membrane proteins laminins have been shown to influence cellular differentiation, adhesion, proliferation, and migration.
• a fertilized oocyte first divides into two cells, followed by another cell duplication to generate a four-cell embryo.
• the embryonic cells are bound together with the help of cell membrane proteins and also the molecules of a new connective tissue (extracellular matrix).
• the first extracellular matrix molecules to appear are basement membrane proteins, such as laminin and proteoglycan.
• the embryonic cells start to differentiate into the three germ cell layers; ectoderm, endoderm and mesoderm, with initiation of morphogenesis.
• the extracellular matrix molecules such as laminins are responsible for interactions with cell surface receptors, thus regulating cell behavior such as adhesion, proliferation, migration and differentiation (Colognato, H., and Yurchenco, P. D. (2000); Form and function: the laminin family of heterotrimers; Dev Dyn 218, 213-234), while other extracellular matrix components such as collagens of types I, II, III or IV primarily serve a mechanical supportive function (Aumailley, M., and Gayraud, B. (1998); Structure and biological activity of the extracellular matrix; J Mol Med 76, 253-265).
• Laminins are large trimeric extracellular matrix proteins that are composed of alpha, beta, and gamma chains. There exist five different alpha chains, three beta chains and three gamma chains that in human tissues have been found in at least fifteen different combinations (Colognato, H., and Yurchenco, P. D. (2000); Form and function: the laminin family of heterotrimers; Dev Dyn 218, 213-234); (Aumailley, M., Bruckner-Tuderman, L, Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J. C, et al.
• laminin-1 that contains alpha-1 , beta-1 and gamma-1 chains
• LN-21 1 is primarily present in BMs of muscle cells and motor neuron synapses, while LN-1 1 1 is restricted to the early embryo and later to certain epithelial cells.
• LN-332 is specifically found in subepithelial BMs and LN-411 is located in subendothelial BMs.
• LN-51 1 is ubiquitously distributed and has been found in BMs of the early embryo and BMs of most adult tissues.
• Laminins are the first ECM proteins cells to be identified in the early embryo, and laminin chains have been detected on the cell surface at the 2-cell stage in mouse embryos.
• Matrigel a complex tumor and BM-like extract obtained from murine Engelbreth-Holm-Swarm (EHS) sarcoma tumor tissues.
• EHS Engelbreth-Holm-Swarm
• Matrigel mainly contains murine LN-111 , type IV collagen, perlecan and nidogen, but also varying amounts of other materials, including growth factors and cellular proteins and, therefore, its composition is undefined and varies from batch-to-batch. This variability can cause irreproducibility of scientific results, and due to the animal origin of the substratum makes Matrigel unacceptable for the expansion and maintenance of hES cells for human cell therapy.
• laminin isoforms are difficult or impossible to extract and purify in native forms from tissues due to extensive crosslinking between ECM proteins, and they can only be produced in minute amounts from cells such as keratinocytes and endothelial cells. Only recently, laminins such as LN-332, LN-41 1 and LN-51 1 have been successfully produced as human recombinant proteins.
• compositions and methods for culturing and growing human embryonic stem cells are provided.
• providing compositions and methods for enabling the proliferation and survival of pluripotent human stem cells in vitro without use of differentiation inhibitory agents such as LIF or feeder cells would be advantageous.
• compositions such as extracellular matrices, and processes for using the same, for culturing human stem cells in vitro in an undifferentiated state.
• laminins provide a defined and suitable extracellular matrix for the growth and proliferation of undifferentiated human embryonic stem cells in vitro. This is absent from feeder cells and/or differentiation inhibitors, or in other words feeder cells and/or differentiation inhibitors are not necessary for the embryonic stem cells to grow and proliferate on the laminin extracellular matrix. Also, it has been found that when pluripotent human embryonic stem cells are cultured on plates coated with recombinant laminin-10 (laminin-51 1 ) in a chemically defined medium, such as an analog of mTeSRI , the cells can proliferate and maintain their pluripotency even in an absence of differentiation inhibitors.
• laminin-10 laminin-51 1
• a chemically defined medium such as an analog of mTeSRI
• Fig. 1 is a microphotograph (phase contrast) of human embryonic stem cells on plates coated with recombinant laminin-10 (laminin-51 1 ) in a chemically defined medium after 105 days of feeder-free culturing.
• Fig. 2 is a photograph of RT-PCR showing the expression of pluripotency markers (Oct4, Nanog), internal control (GAPDH) and differentiation markers (alpha- fetoprotein, brachyury, Sox1 and Pax6) in human embryonic stem cells cultured on laminin-10 (laminin-511 ) in the chemically defined medium for 105 days (LN-51 1 , 20 passages) and on human foreskin fibroblasts (control) in the conventional medium.
• pluripotency markers Oct4, Nanog
• GPDH internal control
• differentiation markers alpha- fetoprotein, brachyury, Sox1 and Pax6
• hGADPH, hOct4, hNanog, hBrach, hAFP, hSoxl and hPax6 stand for GAPDH, Oct4, Nanog, brachyury, alpha-fetoprotein, Sox1 and Pax6, respectively.
• Fig. 3 contains a series of color microphotographs (immunofluorescence) demonstrating human embryonic stem cell self-renewal effect on laminins -511.
• laminin-51 1 in the chemically defined media for 105 days (20 passages)
• human embryonic stem cells continue to express pluripotency markers Oct4 (green) and Sox2 (red) (LN-51 , 20 passages).
• Human embryonic stem cells cultured on human foreskin fibroblasts in the conventional medium also express pluripotency markers Oct4 and Sox2 (control).
• Fig. 4 demonstrates adhesion of human ES cells to different coatings and expression of laminin chains in hES cells.
• Fig. 4a shows Crystal Violet staining of human ES cells adherent to LN-511 and LN-1 1 1.
• Fig. 4b is a graph showing the contact area of ES cells to different adhesive substrata. Values are shown as average relative contact area (compared with the cells plated on poly-D-lysine). Statistical significance (P) was calculated by the Student t-test. Error bars show standard error (SE); number inside each bar shows number of independent measurements.
• SE standard error
• Fig. 4c shows the RT-PCR analysis of total RNA isolated from HS420 cells. Primer sets for all known laminin chains were used.
• Fig. 5 demonstrates integrin receptors on hES cell surface and their role in adhesion to LN-511.
• Ffg. 5a is a graph showing the inhibition of human ES cell adhesion to LN-511 by different anti-integrin antibodies.
• Bars represent (from left to right) ⁇ 1 , ⁇ 6, ⁇ 1 , ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, aV, ⁇ ⁇ 3, ⁇ 2, ⁇ 3, ⁇ 4 (all from Millipore).
• Fig. 5c is a set of immunofluorescence pictures showing integrin a6 co-expression with ⁇ 1 integrin subunit in pluripotent (Sox2-positive) hES cells cultured on LN-511. Magnification x40.
• FIG. 6 shows representative immunostaining analysis, RT-PCR, FACS analysis, real-time quantitative RT-PCR and quantitative Western blot analysis of HS207 cultured on LN-511 in O3 medium and free from any animal derived components H3 medium.
• Fig. 6a shows growth curves for hES cells cultured in O3 medium on LN-511 and Matrigel. The cells were passaged as described below for the long-term experiment. After each TrypLETM Express treatment and subsequent washing only one third of the cells in clumps was used to plate on fresh LN-511 or Matrigel coated dishes. The rest was dissociated into single cell suspension and counted. The experiment was performed in two independent duplicates for each coating.
• Fig. 6b shows immunostaining of HS207 cells with anti-Nanog (labeled as Nanog), anti-Sox2 (Sox2) and anti-Oct4 (Oct4) antibodies after 20 passages (6 months) on LN-511 in O3 medium.
• Right panels are nuclear DAPI staining. Same staining represented with higher magnification. Magnification: x20. Bars: 0.15 mm.
• 6c shows RT-PCR analysis of total RNA isolated from H207 cells grown on feeder cells (depicted as Feeders), on Matrigel after 7 passages in 03 medium (MG, p7 in 03), on LN-511 after 8 passages in H3 medium (LN-511 , p.8 in H3) and on LN-511 after 27 passages in 03 medium (LN-511 , p.27 in 03).
• Primer sets for pluripotency markers Oct4 and Nanog; differentiation markers Brachyury, a-feto protein, Sox1 , and Pax6; and a housekeeping gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed.
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• Bars represent (from left to right) levels of Oct4 and Nanog expression in control HS207 cells cultured on feeder layer (Feeders) taken as standard values to compare, in HS207 cells cultured on Matrigel after 7 passages in 03 medium (MG, p.7 in 03), in HS207 cells cultured on LN-511 after 5 (LN-511 , p.5 in 03), 10 (LN-511 , p.10 in O3), 16 (LN-511 , p.16 in O3), 20 (LN-511 , p.20 in O3) passages in 03 and in the cells on LN-511 after 8 passages in H3 (LN-511 , p.8 in H3). Error bars show 95% confidence interval. Fig.
• FIG. 6e shows expression of pluripotency markers Oct4 and Sox2 in HS207 cells cultured on feeder cells, Matrigel and LN-511 at different time points and in different media as measured by Western blot and quantified by densitometry. Grey bars represent expression levels of Oct4, black ones - Sox2. Abbreviations are similar to Fig. 6d. Error bars represent range.
• Fig. 6f shows FACS analysis of HS207 cells after 25 passages on LN-511 in 03 medium for cell surface marker of pluripotency Oct 4. The percentage of positive cells is listed in parentheses.
• Fig. 7 demonstrates the pluripotency of HS207 cells after extensive passaging on LN-511. Teratomas composed of tissue components of the three germ layers were formed after subcutaneous injection into SCID mice of HS207 cells after culture of 15 passages on LN-511.
• Fig. 7a shows cartilage, HE-staining. Magnification x100.
• Fig. 7b shows developing neural tissue and intestinal endoderm, the goblet cells shown in red, HE-PAS staining.
• Fig. 7c shows developing kidney glomerulus, HE staining.
• Fig. 7d shows retinal pigment epithelium, HE staining. Magnification: x400.
• Fig. 7a shows cartilage, HE-staining. Magnification x100.
• Fig. 7b shows developing neural tissue and intestinal endoderm, the goblet cells shown in red, HE-PAS staining.
• FIG. 7e shows immunostaining of embryoid bodies formed from HS207 cells after 20 passages on LN-5 revealed expression of markers for the three embryonic cell layers: smooth muscle actin (depicted as SM Actin), Nestin (Nestin), MAP-2 (MAP-2) and a- fetoprotein (AFP). Bottom panels are nuclear DAPI staining.
• smooth muscle actin depicted as SM Actin
• Nestin Nestin
• MAP-2 MAP-2
• AFP a- fetoprotein
• Fig. 8 shows immunostaining analysis of different hES cells grown on LN- 511.
• Fig. 8 shows H1 and H9 cells after 5 passages (1 month) on LN-511 in O3 medium expressed markers of pluripotency Oct4(green), Nanog(green), and Sox2(red).
• DAPI staining is in blue.
• Fig. 9 shows G-banding chromosome analysis of HS207 cells after 20 passages on LN-51 1.
• Fig. 10 shows FACS analysis of HS207 cells after 20 passages on LN-51 1 in 03 medium for cell surface markers of pluripotency (SSEA-4, Tra1 -60, Tra1 -81 ) and for cell surface marker of differentiation SSEA-1. The percentages of SSEA-4, Tra1-60, and Tra1 -81 positive cells are listed in parentheses.
• Fig. 11 shows FACS analysis of HS207 cells after 10 passages on Matrigel in O3 medium for markers of pluripotency Oct-4 and SSEA-4. The percentage of positive cells is listed in parentheses.
• Fig. 12 is a graph showing levels of spontaneous differentiation in LN-51 1 , Matrigel and feeder HS207 cell cultures.
• Real-time quantitative RT-PCR analysis was used to compare number of mRNA transcripts of differentiation markers Pax6, Sox17, and Sox7 in HS207 cells cultured on Matrigel in O3 medium(white bars), on LN-51 1 in O3 medium (grey bars) and on human foreskin fibroblast layer in a serum replacement based medium (black bars). Error bars show 95% confidence interval.
• Fig. 13 is a set of pictures showing early stages of derivation of the hESC line HS588 on LN51 1 in O3 medium.
• Fig. 13a shows morphologically typical hESC growing out from the inner cell mass of a day six blastocysts four days after mechanical isolation of the inner cell mass and plating on LN5 1.
• Fig. 13b shows immunostaining of HS588 cells with anti-Nanog antibodies at passage 2 on LN-51 1 in O3 medium.
• Fig. 13c shows immunostaining of the same cells with anti-Oct4 antibodies.
• Fig. 14 shows the characterization of human recombinant LN-51 1 used in the article using 3-8 % gradient SDS-PAGE under reducing and non-reducing conditions. The protein was visualized using Sypro Ruby protein staining.
• compositions and processes for culturing human stem cells in vitro in an undifferentiated state are disclosed.
• human embryonic stem cells proliferated and maintained their pluripotency when cultured on plates coated with recombinant laminin-10 (laminin-51 1 ).
• a feeder-free and xeno-free hES cell culture system contains human albumin purified from plasma and a human protein mixture for culture dish coating.
• the human proteins cell culture dish coating is reported to contain IV collagen, vimentin, fibronectin and human laminin, which has been shown to be a mixture of degraded proteins from human placenta. It contains fragments of several laminin types and frequently also other BM proteins and fibronectin. Therefore, these hES cell conditions are variable, cannot be considered chemically defined, and the component(s) of the matrix providing self-renewal are unknown.
• the O3 medium was a variant of the commercially available chemically defined mTeSRI medium with bovine serum albumin as the only animal derived component.
• the H3 medium was a variant of chemically defined and xeno-free TeSR1 medium containing human serum albumin.
• Applicants dialyzed the protein solution using 12-14KDa dialysis membranes. To passage cells free from any animal-derived components, TrypLETM Express enzyme was used. Control cells were cultured on Matrigel in O3 medium or on a layer of feeder cells in a serum replacement based medium. Applicants studied the adhesion of hESC cells to different ECM proteins. LN-51 1 provided the largest cell contact areas with the substratum. Human induced pluripotent stem (iPS) cell lines also self-renewed on LN-51 1. The adhesion was dependent on ⁇ 6 ⁇ 1 integrin, which has been showed to be most abundant on hES cell surfaces.
• iPS Human induced pluripotent stem
• LN-511 was expressed in hES cells and as a part of hES cell environment in feeder-dependent cell cultures and in the early embryo could be a natural substratum for hES cell adhesion, migration and proliferation.
• pluripotent human embryonic stem cells are cultured on plates coated with recombinant human laminin-10 (laminin-511 ) in chemically defined medium, the cells proliferate and maintain their pluripotency for at least 105 days (20 passages) (Figs. 1-3).
• pluripotency markers such as Oct4, Sox2 and Nanog, and the proliferation rate, also remained stable.
• self-renewal refers to the ability of the stem cell to go through numerous cycles of cell division and remain undifferentiated (i.e. pluripotent). Pluripotency itself refers to the ability of the stem cell to differentiate into any cell type.
• proliferation refers to the ability of the stem cell to divide. Survival refers to the ability of the stem cell to live, whether differentiated or undifferentiated, and does not require the stem cell to maintain its ability to divide or to differentiate.
• Human embryonic stem cells (two lines were used: HS420 and HS207, both kindly provided by Prof. Hovatta, Karolinska University Hospital Huddinge, Karolinska Institute, Sweden) were cultured on plates coated with recombinant laminin-10 (laminin-511 ) in the chemically defined medium, analog of mTeSRI .
• the medium was prepared as described in (Ludwig, T.E., Bergendahl, V., Levenstein, M.E., Yu, J., Probasco M.D. and Thomsom, J.A. (2006); Feeder-independent culture of human embryonic stem cells; Nat Methods 8, 637-646) with several exceptions.
• recombinant human FGF basic (R(5)DSvstems) was used instead of zbFGF and albumin from bovine serum (SIGMA-Aldrich, B4287) was used instead of BSA fraction V.
• Insulin-Transferrin-Selenium Supplement Insulin-Transferrin-Selenium Supplement added in already made medium was used as a source of the elements instead of the method described in the article.
• the human embryonic stem cells were passages in clumps at 4-6 days intervals by exposure to TrypLE Express (GIBCO). The cells were subjected to the enzyme for 2 minutes at room temperature, then washed 2 times with the medium, followed by gentle scraping to collect. Big clumps of the cells were broken by gentle pipetting and 1 :3 passaged.
• Control human embryonic stem cells were maintained on human foreskin fibroblasts in the conventional medium as described in (Inzunzaa, J., Gertow, K., Stromberg, ⁇ ., ⁇ ., Matilainen, E., Blennow, E., Skottman, H., Wolbank, S., Ahrlund- Richter, L. and Hovatta, O. (2005); Derivation of Human Embryonic Stem Cell Lines in Serum Replacement Medium Using Postnatal Human Fibroblasts as Feeder Cells. Stem Cells 2005;23:544-549). The cells were mechanically passaged by cutting the colony to eight pieces using a scalpel under the stereo microscope. Mechanical splitting was carried out at 6-day intervals. Nondifferentiated cells, as judged by morphology, were chosen for each further passage.
• 96-well tissue cell culture plates (Sarstedt) were coated overnight at 4°C by sterile solutions of extracellular matrix proteins: murine laminin-111 (Invitrogen), human recombinant laminin-332, human recombinant laminin-411 (Kortesmaa, J., Yurchenco, P., and Tryggvason, K. (2000); Production, purification, and interactions with integrins. J Biol Chem 275, 14853-14859, U.S.
• Patent 6,638,907 human recombinant laminin-511 (Doi, M., Thyboll, J., Kortesmaa, J., Jansson, K., livanainen, A., Parvardeh, M., Timpl, R., Hedin, U., Swedenborg, J., and Tryggvason, K. (2002); Production, purification, and migration-promoting activity on vascular endothelial cells. J Biol Chem 277, 12741-12748; U.S.
• Patent 6,933,273 all in concentration 30 ug/ml (5 ug/mm 2 ), growth factor-depleted MatrigelTM (1 :30) (BD Biosciences), bovine gelatin 1 mg/ml (Sigma), 0.1 mg/ml poly-D-lysine (Sigma).
• Attachment assay was performed as described ([Extracellular Matrix Protocols, 2000). Briefly, MaxiSorp 96-well plates (Nunc) coated by extracellular matrix proteins as described above and blocked by 1 % heat-denatured BSA solution. Undifferentiated embryonic cells were plated at cell density of 800 cell / mm 2 upon extracellular matrix-coated plates and were left to adhere for 1 hour at 37°C. Nonadherent cells were washed away, and adherent cells were fixed for 20 min by 5% glutaraldehyde, stained by 0.1% Crystal Violet.
• PCR reactions were run for 30 cycles (including those GADPH PCRs which are shown on pictures) and were performed in 20 ⁇ under standard conditions using 1 U of Taq DNA Polimerase Recombinant (Invitrogen). The PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide.
• RNA sample For each RNA sample, RT-PCR without reverse transcriptase was performed to confirm that no genomic DNA was isolated.
• Morphology of human embryonic stem cells cultured on Laminin -51 1 was very similar to that found for human embryonic stem cells cultured on MatrigelTM (Bendall, S.,C, Stewart, ⁇ ., ⁇ ., Menendez, P., George, D., Vijayaragavan, K., Werbowetski-Ogilvie, T., Ramos-Mejia, V., Rouleau, A., Yang, J., Bosse, M., Lajoie, G. and Bhatia, M. (2007); IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.
• RT-PCR markers Pluripotency markers Oct4 and Nanog were expressed at same extent by human embryonic stem cells cultured on laminins -551 in the chemically defined medium for 105 days, as pluripotent embryonic stem cells cultured on human fibroblast foreskin in the conventional medium. See Fig. 2.
• Cells were fed once a day with fresh medium pre-warmed in an incubator for 1 hour, except for the first day after a passage when only a few drops of fresh medium were added. Cells were routinely passed once in 6-7 days by exposure to TrypLETM Express (GIBCO Invitrogen Corporation, Paisley, Scotland) for 1.5 minutes at room temperature. Then they were washed two times on the dish with the medium, gently scraped, pipetted to break into small pieces (not a single cell suspension) and plated in 1 :2 or 1 :3 ratio (up to 1 :6 ratio if a large number of cells was needed). Control cells of the same line were cultured on Matrigel in O3 medium and on human foreskin fibroblasts. Laboratory dishes were coated. Prior to use, dishes were pre-warmed in an incubator for 1 hour and then carefully washed twice with the pre-warmed medium.
• Stock A was prepared by adding 165 mg of thiamine and 50 mg of reduced glutathione to 500 ml of distilled water as described in Ludwig, Nat. Methods 3:637-646 (2006), but without L-ascorbic acid.
• the distilled water was purchased from GIBCO Invitrogen Corporation. Then the solution was filtered (0.22 pm filter), aliquoted and frozen at -20°C.
• Stock B was prepared as described in Ludwig, but without selenium, insulin and holo-transferrin. Then 6 pg of Phenol Red was added, carefully stirred and filtered. Stock B could be stored at +4°C up to 2 months.
• TGF- i transforming growth factor
• GABA ⁇ - aminobutyric acid
• LiCI LiCI
• D-MEM/F12 medium was supplemented with 20 ml of Stock B, 200 ⁇ of TGF- ⁇ stock, 13 ⁇ of pipecolic acid stock, 200 ⁇ of GABA stock, 200 ⁇ of LiCI stock, 1 ml of MEM non-essential amino acid solution (GIBCO Invitrogen Corporation), 1 ml of 200mM L-glutamine solution (GIBCO Invitrogen Corporation) and 2 ml of insulin-transferrin-selenium supplement (GIBCO Invitrogen Corporation). To compensate the salt balance and to adjust pH of the medium 145 mg of NaCI and 56 mg of NaHCO 3 were added.
• O3 medium could be stored at +4°C up to 1 month. Before use the medium was supplemented with 96 ng/ml of recombinant human FGF basic (R@D Systems Europe LTD, Abingdon, England) and 40 pg/ml of ascorbic acid (SigmaAldrich).
• TGF- ⁇ Stock of TGF- ⁇ was prepared as described in Ludwig, but dialyzed human albumin instead of bovine albumin was used. All other stocks were prepared as described above.
• H3 medium was mixed was described for 03, but NaCI was not added at all.
• the medium was supplemented with 96 ng/ml of carrier free recombinant human FGF basic (R@D Systems Europe LTD, Abingdon, England) and 40 pg/ml of ascorbic acid (SigmaAldrich).
• Human recombinant LN-511 available from BioLamina, AB, Sweden, was produced in human embryonic kidney cells (HEK293; ATCC CRL-1573) sequentially transfected with full-length laminin ⁇ 1 , ⁇ 1 and ⁇ 5 constructs.
• HEK293 human embryonic kidney cells
• DMEM Dulbecco's modified Eagle's medium
• GlutaMax I GlutaMax I
• GIBCO Invitrogen Corporation GlutaMax I and 4.5 g/l glucose
• the LN-5 1 molecules were affinity-purified using anti-FLAG matrix (Sigma), and then characterized using 3-8% (Fig. 14) and 4-15% gradient SDS-PAGE under reducing and nonreducing conditions.
• the proteins were visualized using Sypro Ruby (Bio-Rad) protein staining and immunostaining of the chains on polyvinylidene difluoride membranes. To further characterize the protein, Western blot analysis with antibodies against the laminin ⁇ 5, ⁇ 1, and ⁇ 1 chains was performed. The preparations contained all three chains of the right size. Human recombinant Laminin-411 was produced in a similar fashion as LN-511. All other ECM proteins were obtained as described previously.
• Adhesion-blocking assays were performed. Briefly, plates were coated by LN-511 and blocked by 1 % heat-denatured BSA solution. ES single-cell suspension was incubated with function-blocking anti-integrin antibodies (concentration as recommended by supplier) for 30 minutes, plated on LN-511 -coated plates and allowed to adhere for 1 hour at 37°C. Non-attached cells were removed, the remaining cells were fixed, and adherent cells were fixed for 20 minutes by 5% glutaraldehyde, washed and stained by 0.1% Crystal Violet. After one hour, Crystal Violet was extracted from cells by 10% acetic acid and quantified by measuring optical density at 570 nm.
• the assay was designed to identify integrin receptors that are expressed in sufficient amounts to retain cells attached to the surface coated with anti-integrin- specific antibody.
• MaxiSorp 96-well plates (Nunc) were coated with purified anti- integrin antibodies at a concentration of 10 pg/ml at +4°C overnight and later washed and blocked with 1 % BSA solution.
• ES cells were plated on antibody-coated plates and allowed to adhere for 1 hour at 37°C. Nonattached cells were removed, and the remaining cells were fixed, stained, and quantified as described above. Error bars show SEM.
• RNA was isolated using Absolutely RNA Microprep Kit (Stratagene, La Jolla CA, www.stratagene.com) according to the manufacturer's instructions.
• cDNA was synthesized using 0.2 pg of total RNA in 20 ⁇ reaction mixture, containing oligo(dT)12-18 primers and Superscript II reverse transcriptase (GIBCO Invitrogen Corporation), according to the manufacturer's instructions.
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• Amounts of cDNA yielding equivalent amount of GAPDH PCR product were used for subsequent PCR reactions.
• cDNAs were amplified using primers described in Table 2.
• primers were used. All PCR reactions were run for 30 cycles (including those GAPDH PCRs which are shown in the figures) and were performed in 20 ⁇ under standard conditions using 1 U of Taq DNA Polymerase Recombinant (GIBCO Invitrogen Corporation). The PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide. For each RNA sample, RT-PCR without reverse transcriptase was performed to confirm that no genomic DNA was isolated.
• ES cells were cultured and fixed in 8- well slide chambers (BD Biosciences) or 96-well plate wells by 4% paraformaldehyde, permeabilized by 0.1 % Triton-X and blocked by 10 % bovine fetal serum (GIBCO Invitrogen Corporation) in phosphate-saline buffer (PBS) containing 0.1 % Tween-20 (Sigma-Aldrich, St. Louis, MO) for one hour. Incubation with primary antibody was performed for 1.5 hours at room temperature. Incubation with secondary antibody and 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was performed for 40 minutes.
• PBS phosphate-saline buffer
• Tween-20 Sigma-Aldrich, St. Louis, MO
• specimens were washed with 0.1 % Tween-20 in PBS buffer three to five times. Specimen were preserved in fluorescence mounting medium (Dako, Glostrup, Denmark), and observed under a fluorescence microscope (Leica, Heerbrugg, Switzerland).
• Real-time quantitative RT-PCR Taqman assays were performed using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA). All reactions were done in quadruplicates with the use of pre-developed gene expression assay mix (Applied Biosystems) containing primers and a probe for the mRNA of interest. Additional reactions for each experiment included pre- developed gene expression assay mix for GAPDH for normalizing the RNA input. All data was analyzed with 7300 System SDS Software v 1.4.
• hES cells were collected, counted and pelleted by centrifugation, mixed with non-reduced SDS-PAGE sample buffer to equal concentration of 2000 cells/ ⁇ and sonicated 5 times for 15 seconds. Gradient 4-12% gels were used for SDS electrophoresis and the proteins were transferred to PVDF membranes. Membranes were blocked by 5% milk solution in PBS-0.1 % Tween buffer for 2 hours. Primary antibody against Oct4 and Sox2 (both from Millipore) in 5% milk solution in PBS with 0.1% Tween buffer were incubated with the membranes overnight at +4°C.
• Metaphase spreads were prepared on glass microscope slides and G-banded by brief exposure to trypsin and stained with 4:1 Gurr's/Leishmann's stain (Sigma-Aldrich Co.). A minimum of 10 metaphase spreads were analyzed and an additional 20 were counted.
• Teratoma formation experiments were done by implantation of approximately 106 cells beneath the testicular capsule of a young (7 weeks old) severe combined immunodeficiency (SCID) mouse. Three animals per each cell line were used. Teratoma growth was determined by palpation every week, and the mice were sacrificed 8 weeks after the implantation. The teratomas were fixed, and sections were stained with hematoxylin and eosin (HE) or with hematoxylin, eosin and PAS (HE-PAS). The presence of tissue components of all three embryonic germ line layers was shown, as analyzed from the stained sections. All animal experiments were performed at the infection-free animal facility of the Karolinska University Hospital in accordance with ethical committee approval.
• SCID severe combined immunodeficiency
• ES cells were dissociated from LN-511 coated cell culture dishes as described above for passaging them, broken into pieces and cultured in suspension 96-well plates (Sarstedt).
• the medium used for this was Knockout DMEM (GIBCO Invitrogen Corporation) supplemented with 2 mM L-glutamine, 20% fetal calf serum (GIBCO Invitrogen Corporation), 0.1 mM ⁇ -mercaptoethanol (GIBCO Invitrogen Corporation) and 1 % non-essential amino acids (GIBCO Invitrogen Corporation).
• the embryoid bodies were transferred into gelatin coated tissue cell culture 96-well plates (Sarstedt), cultured for 1 week, then fixed, stained with antibodies against markers of all three embryonic germ line layers (smooth muscle actin, Nestin, MAP-2 and a-fetoprotein, all three antibodies were from Millipore) and analyzed as described above for immunofluorescence.
• the average contact area of an adherent human ES cell grown on LN-5 was about 1.6 times higher than that of cells plated on Matrigel and about 1.2 times higher than that of cells plated on LN-332 (Fig. 4b). Embryonic stem cells spreading on other coatings tested were significantly less than that on LN-511.
• the HS420, HS207 and HS401 lines had undergone 28, 27 and 25 passages in O3 medium, respectively (5-6 months in culture).
• Karyotypes were normal for all three lines after 20 passages (Fig. 9).
• the HS420 and HS207 lines have, thus far, proliferated for 29 and 27 passages in H3, respectively (5 months).
• Immunofluorescence and RT-PCR analyses revealed that hES cells maintained high expression level of pluripotency markers, such as Oct4, Nanog, and Sox2 (Figs. 6b and 6c).
• HS207, HS420 and HS401 cells cultured for 15, 20 and 20 passages on LN-51 1 in O3 medium and HS207 cells cultured for 23 passages in H3 medium formed teratomas after being grafted into the testes of severe combined immunodeficient mice (SCID). Analysis of the stained sections confirmed the ability of the cells to differentiate into cells of all three germ lineages of the human embryo (Figs. 7a-7d). To further examine the developmental potential, the cells of all three hES lines after 20 passages on LN-51 1 in O3 medium were examined in vitro by the formation of embryoid bodies and subsequent immunostaining on markers of all three embryonic germ layers.
• the staining revealed expression of mesoderm (smooth muscle actin), ectoderm (Nestin and MAP-2) and endoderm (a-fetoprotein) markers (Fig. 7e), thus providing additional evidence of pluripotency of the cells.
• H1 and H9 hES lines cultured well characterized and widely used H1 and H9 hES lines cultured on the protein in O3 or mTeSRI medium.
• the H1 and H9 cells had phenotype and proliferation rate similar to that of HS207, HS420 and HS401 cells under the same conditions.
• Immunofluorescence analysis revealed that after 5 passages (1 month) in culture on LN-511 , the H1 and H9 cells maintained expression of pluripotency markers, such as Oct4, Nanog, and Sox2 (Fig. 8).
• LN-51 1 provided an artificial niche for supporting survival and self-renewal of human ES cells in culture in a xeno-free environment for at least 20 passages, or for 4 months. Importantly, the cells did form teratomas containing cell lineages of all three germ lineages of the human embryo after being cultured on LN-51 1 for that long period. Since there is a great need for a chemically defined xeno-free feeder free culture systems for hES cells, the system described here may provide a solution for that problem.
• LN-51 1 is likely to be part of the natural niche for stem cells in the human embryo, as this protein has been detected in the inner cell mass of the blastocysts which is the natural origin of human ES cells. Furthermore, LN-511 is expressed in colonies of human embryonic stem cells cultured in vitro on feeder cells. Moreover, human ES cells express LN-51 1 themselves (Fig. 4c), and the protein promotes ES cells assembly, which is crucial for their self-renewal on a feeder layer. Since LN-511 is a part of the natural environment of hES cells, it can provide a biologically relevant coating matrix for self-renewal of human ES cell in vitro.
• mice It was recently reported that hES cells stayed pluripotent on several recombinant human laminins, such as LN- 1 11 , LN-332 and LN-51 1 for 96 hours. However, a previous study with mouse ES cells showed that it is important to culture the cells longer in order to demonstrate the capacity of the matrix molecules for ES cells self-renewal. In that study, mouse ES cells survived and proliferated on both LN-51 1 and LN-332 for at least 169 days, but only mouse ES cells cultured on LN-511 were able to generate germ-line competent chimeric mice.
• hES cells tended to form monolayers after being passaged in clumps to new LN-511 coated plates. This suggests that LN-511 could normally provide human ES cells with a migration potential, thus avoiding formation of dense multilayer clusters with poor transfer of soluble nutrients and growth factors. Availability of soluble factors for hES cells cultured on LN-511 can help to avoid spontaneous differentiation and mortality, which usually appears in the middle of overgrown ES cell colonies when cultured on feeders or Matrigel. Monolayers of hES cells could also be beneficial for development of differentiation procedures, since equal availability of soluble factors could create more homogeneous populations of differentiated cells.
• Applicants showed that the mechanism of human ES cells attachment to LN-511 was also strongly dependent on binding to ⁇ 6 ⁇ 1 integrin receptor (Fig. 5a). Moreover, Applicants observed that antibodies against ⁇ 1 integrin supported stronger attachment of the human ES cells than ⁇ 2, ⁇ 3 or ⁇ 4 integrins, emphasizing the pivotal role of ⁇ 1 containing integrins in cell adhesion (Fig. 5b). Recently, it has been shown that ⁇ 6 ⁇ 1 is the most abundant integrin isoform on a hES cell surface24.
• LN- 511 human ES cells abundantly expressing ⁇ 6 ⁇ 1 integrins can attach fast and efficiently migrate on a LN- 511 coated surface that, in turn, facilitates their self-renewal.
• LN- 511 could be to provide to human ES cells focal adhesion contacts to the surface and enable mobility on it.
• LN-511 expression is not restricted to the early embryos, but found in basement membranes of many adult tissues, supports that notion.
• a chemically defined substratum is a major advantage compared with using feeder cells for the same purpose, because feeder cells can vary in their production of bioactive molecules, such as cytokines, growth factors and other proteins are largely unknown, and they can also pose a risk for microbial and viral contamination, as well as batch-to-batch variability in the results.
• the LN-51 1 based xeno-free and feeder-free human ES cell culture system described in this study can be a significant solution for this problem. In most countries, regulatory authorities apply strict rules to any reagents that are intended for use in human therapy.
• human recombinant LN-51 1 is a chemically defined protein produced in human cells, it is feasible to establish a production protocol that can yield the protein suitable for the development of human cells for cell therapy. Manufacturing can be easily taken into a good manufacturing practice (GMP) laboratory.
• GMP manufacturing practice
• the TeSR1 medium was formulated for use with Matrigel. It contains many growth factors in very high concentrations. Since LN-51 1 provided better spreading of hES cells and high average expression of the main pluripotency markers, it opens up for a possibility to optimize the medium composition in order to decrease the doses of the growth factors, especially bFGF. Unlike Matrigel, LN-51 1 is a defined homogeneous protein molecule that is stable from batch-to-batch coating. The use of a more defined environment to culture hES cells in vitro can help to develop and understand the molecular mechanism of differentiation and provide more controllable conditions for design of differentiation pathways for human ES cells in vitro.

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