WO2006070370A2 - Stem cells culture systems - Google Patents

Stem cells culture systems Download PDF

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WO2006070370A2
WO2006070370A2 PCT/IL2005/001397 IL2005001397W WO2006070370A2 WO 2006070370 A2 WO2006070370 A2 WO 2006070370A2 IL 2005001397 W IL2005001397 W IL 2005001397W WO 2006070370 A2 WO2006070370 A2 WO 2006070370A2
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cells
culture system
human
culture
serum
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PCT/IL2005/001397
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English (en)
French (fr)
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WO2006070370A3 (en
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Etti Ben Shushan
Shelly Tannenbaum
Pavel Itsykson
Eyal Banin
Benjamin Reubinoff
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Hadasit Medical Research Services & Development Limited
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Priority to EP05821535.1A priority Critical patent/EP1844136B1/en
Priority to ES05821535.1T priority patent/ES2525684T3/es
Priority to US11/794,262 priority patent/US20090104695A1/en
Priority to EP11174158.3A priority patent/EP2410044B1/en
Publication of WO2006070370A2 publication Critical patent/WO2006070370A2/en
Publication of WO2006070370A3 publication Critical patent/WO2006070370A3/en
Priority to US11/730,560 priority patent/US8597947B2/en
Priority to US13/005,978 priority patent/US9005965B2/en

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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the invention relates to stem cells (SC) in particularly to methods and systems for handling human embryonic stem cells (hESC).
  • SC stem cells
  • hESC human embryonic stem cells
  • Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.
  • hESC Human embryonic stem cells
  • ES cells are unique stem cells since they can self-renew infinitely in culture, and since they have a remarkable potential to develop into extraembryonic lineages as well as all somatic cells and tissues of the human body 1 ' 2 .
  • hESC Given the unique properties of hESC, they are expected to have far- reaching applications in the areas of basic scientific research, pharmacology, and regenerative medicine.
  • Human ES cell lines can provide a powerful in vitro model for the study of the molecular and cellular biology of early human development, for functional genomics, drug screening, and discovery. They may serve for toxicology and teratogenicity high throughput screening.
  • hESC can self-renew indefinitely and can differentiate into any cell type, they can serve as a renewable, unlimited donor source of functionally mature differentiated cells or tissues for transplantation therapy.
  • transplanted genetically- modified hESC can serve as vectors to carry and express genes in target organs in the course of gene therapy.
  • hESC While the promise of hESC for basic scientific research pharmacology and regenerative medicine is remarkable, the exploitation of hESC for most applications depends upon further development. Improved control of the growth of undifferentiated hESC, the development of bulk feeder-free cultures of undifferentiated cells, the development of animal-free culture systems, and the development of methods and tools which direct the differentiation and generate pure cultures of mature functional cells of a specific type are required.
  • undifferentiated hESC 1"4 At present, few culture systems are most commonly used to propagate undifferentiated hESC 1"4 .
  • undifferentiated hESC are cultured in serum-containing medium as colonies, upon a layer of fibroblast feeder cells (of mouse 1 ' 2 or human origin 5 ' ⁇ ). It is possible to remove all animal products from this culture system and replace them with those from a human source 6 . It was found that in this system the cells are propagated as clumps on a low scale which does not allow cloning 2 .
  • KO knockout
  • KOSR knockout serum replacement
  • FGF2 FGF2
  • This system allows cloning of undifferentiated hESC, although at a low efficiency 3 .
  • Undifferentiated cells are cultured as flat colonies and may be propagated as small clusters or single cells (by using trypsin 7 ).
  • Another alternative culture system for use in the proliferation of undifferentiated growth of hESC comprises a culture matrix comprising extracellular matrix (ECM) prepared from feeder cells and a conditioned medium being preconditioned by feeder cells.
  • ECM extracellular matrix
  • the suggested leading cells in the feeder cells include primary mouse embryonic fibroblasts (PMEF) a mouse embryonic fibroblast cell line (MEF) murine foetal fibroblasts (MFF) human embryonic fibroblasts (HEF) human foetal muscle (HFM) human foetal skin cells (HFS) human adult skin cells, human foreskin fibroblasts (HFF) 10 human adult Fallopian tubal epithelial cells (HAFT) or human marrow stromal cells (HMSC).
  • PMEF primary mouse embryonic fibroblasts
  • MFF mouse embryonic fibroblast cell line
  • MFF murine foetal fibroblasts
  • HMF human foetal muscle
  • HFS human foetal skin cells
  • HFF human adult skin cells
  • HFF human
  • Undifferentiated propagation may be accomplished with the KO serum- free culture system without the use of feeders by plating and growing colonies on extracellular matrices (ECM) within a feeder-conditioned KO medium supplemented with KOSR and FGF2 4 .
  • ECM extracellular matrices
  • feeder conditioning may be replaced by substituting the medium with high concentrations of FGF2 and noggin 12 .
  • feeder conditioning was replaced by transforming growth factor ⁇ 1 and human LIF (in addition to FGF2) and growing the cells on human fibronectin .
  • undifferentiated propagation of hESC colonies, in the absence of feeders' was reported with a chemically defined medium without serum replacer, supplemented with activin or nodal plus FGF2 .
  • hESC culture systems do not allow the propagation of pure populations of undifferentiated stem cells and their use always involves some level of background differentiation.
  • the stem cells most commonly follow a default pathway of differentiation into an epithelial cell type that grows either as a monolayer of flat squamous cells or form cystic structures. Most probably, this form of differentiation represents differentiation of hESC into extraembryonic endoderm 9 .
  • the present invention provides a cell culture comprising cells obtained from human umbilical cord tissue, the human umbilical cord derived cells being capable of maintaining stem cells (SC) in an undifferentiated state when co-cultured therewith.
  • the human umbilical cord derived cells are preferably used as feeder cells in SC cultures.
  • the invention also provides a first culture system for maintenance of SC in an undifferentiated state, the culture system comprising feeder cells expanded from human umbilical cord cells, human embryonic fibroblast cells (HEF) and a combination of same.
  • the culture system comprises the human umbilical cord derived feeder cells of the invention.
  • an undifferentiated pluripotent human embryonic SC culture obtained by incubating a cluster of cells from inside a blastocyst with the first culture system of the invention.
  • the invention also provides a method for maintaining SC in an undifferentiated state, the method comprising incubating said cells with a culture system comprising feeder cells expanded from human umbilical cord cells, human embryonic fibroblast cells (HEF) or a combination of same.
  • a culture system comprising feeder cells expanded from human umbilical cord cells, human embryonic fibroblast cells (HEF) or a combination of same.
  • the use of feeder cells expanded from human umbilical cord derived cells, human embryonic fibroblast cells (HEF) and a combination of same for the preparation of a culture system for maintenance of SC in an undifferentiated state also forms part of the invention.
  • the invention provides a further, second, culture system for inhibiting or preventing differentiation of SC to extraembryonic cells, the culture system comprising nicotinamide (NA) or a derivative of NA having an inhibitory effect on differentiation of stem cells to extraembryonic cells similar to that of NA.
  • NA nicotinamide
  • a human embryonic SC culture essentially free of extraembryonic cells is also provided in the context of this aspect of the invention, the SC culture being obtained by incubating a cluster of cells from inside a blastocyst with a culture system comprising said NA or derivative thereof.
  • a method for inhibiting or preventing differentiation of stem cells to extraembryonic cells comprises incubating said stem cells in a culture system comprising NA or a derivative of NA having an inhibitory effect on differentiation of stem cells to extraembryonic cells similar to that of NA.
  • NA or a NA derivative having an inhibitory effect on differentiation of SC to extraembryonic cells similar to that of NA for the preparation of a culture system for inhibiting or preventing differentiation of SC to extraembryonic cells.
  • a further, third, culture system a humanized culture system for maintenance of SC in an undifferentiated state, the culture system comprising an animal free basic stem cell culture medium and humanized serum replacement substitute.
  • an undifferentiated human embryonic SC culture obtained by incubating a cluster of cells from inside a blastocyst with the humanized culture system comprising the animal free stem cell basic culture medium and a humanized serum replacement substitute.
  • a method of maintaining stem cells in an undifferentiated state comprises incubating said cells with a culture system comprising animal free stem cell basic culture medium and humanized serum replacement substitute.
  • a culture system for maintenance SC in an undifferentiated state comprising NeurobasalTM medium.
  • SC culture in an undifferentiated state, the SC culture being obtained by culturing a cluster of cells from inside a blastocyst with a culture system comprising NeurobasalTM medium.
  • a method for maintaining a culture of SC in an undifferentiated state comprising incubating said cells with a culture system comprising NeurobasalTM medium as well as the use of Neurobasal medium for the preparation of a culture system for maintaining a suspension of stem cells in an undifferentiated state.
  • the SC may be maintained in the NeurobasalTM-based culture system in the form of a suspension as well as in a monolayer (flat colonies).
  • the NeurobasalTM medium is supplemented with N2 supplement or an N2 like supplement as defined below.
  • Figures 1A-1D - are phase contrast images of cord fibroblasts primary culture (Fig. IA), human embryonic fibroblasts (Fig. IB), fibroblasts derived from umbilical cord (Fig. 1C) and from foreskin (Fig. ID).
  • Figure 2A-2F are immunofmorscent images of umbilical cord foreskin, and human embryonic fibroblasts stained by anti-vimentin antibody (Figs. 2A, 2C and 2E, respectively) and the corresponding DAPI nuclear counter staining (Figs. 2B, 2D and 2F) showing that the human feeders express vimentin.
  • Figure 3 is a bar graph showing FACS analysis of the percentage of feeders expressing CD44 and that are immunoreactive with anti-fibroblast antibody indicating that a high percentage of the feeders that are derived from the three sources express CD44 and are immunoreactive with anti-fibroblast antibody.
  • Figures 4A-4B - are representative analysis of one metaphase plate of human embryonic fibroblasts (Fig. 4A) and foreskin (Fig. 4B) showing that the human feeders have a normal karyotype.
  • Figure 5A-5C - are phase contrast images of colonies of undifferentiated hESC that are cultured on three types of human feeders, on umbilical cord derived feeders (Fig. 5A), human embryonic fibroblasts (Fig. 5B) and on foreskin derived feeders (Fig. 5C)
  • Figure 6A-6L are representative FACS histograms of marker expression by hESC cultured on the three feeder fibroblast types, including expression of
  • FIGs. 6A-6D by hESC on human embryonic fibroblasts (Fig. 6E- 6H 5 respectively) or by hESC on foreskin (Fig. 6I-6L, respectively).
  • Figure 7A-7C are immunofluorescent images of hES colonies expressing AP 5 when cultured on foreskin derived feeder cells (Fig. 7A), on umbilical cord derived feeder cells (Fig. 7B) and on human embryonic fibroblast cells (Fig. 7C).
  • Figure 8A-8F - are immunofluorescent images (Figs. 8A, 8B and 8C) and the corresponding DAPI nuclear counter staining (Figs. 8D, 8E and 8F) of hESC colonies expressing Oct4 when cultured on human embryonic fibroblast cells (Figs. 8A and 8D, cultured for 6 weeks), on foreskin derived feeders (Figs. 8B and 8E, cultured for 1 week) and on umbilical cord derived feeder cells (Fig. 8C and 8F, cultured for 10 weeks).
  • Figure 9 - is a bar graph representing FACS analysis of the percentage of hESC cultured on two independent cord derived feeder cell lines (CORDl and CORD2), and expressing the indicated markers of undifferentiated pluripotent stem cells at early (1-5) and late (6-10) passage levels, showing that the percentage of hESC expressing these markers is stable during propagation, as determined after 5, 8, 4 and 9 weeks of culture (5 W, 8 W, 4W and 9W).
  • CORDl and CORD2 independent cord derived feeder cell lines
  • Figure 10 - is a bar graph representing FACS analysis of the percentage of hESC cultured on two independent foreskin-derived feeder cell lines (OR2 and OR4), and expressing the indicated markers of undifferentiated pluripotent stem cells at early (1-5) and late (6-10) passage levels, showing that the percentage of hESC expressing these markers is stable during propagation as determined after 3, 6, and 8 weeks of culture (3 W, 6W and 8W).
  • Figure 11 - is a bar graph representing FACS analysis of the percentage of hESC cultured on two independent human embryonic fibroblast feeder cell lines (HEFl and HEF2), and expressing the indicated markers of undifferentiated pluripotent stem cells at early (1-5) and late (6-10) passage levels, showing that the percentage of hESC expressing these markers is stable during propagation, as determined after 2, 5 and 10 weeks (2W 5 5 W and 10W).
  • Figure 12 - is a bar graph representing analysis of the percentage of hESC cultured on two independent cord derived feeder cell lines (CORDl and
  • Figure 13 - is a bar graph representing analysis of the percentage of hESC cultured on two independent foreskin-derived feeder cell lines (OR2 and OR4), and expressing Oct 4 at early (1-5) and late (6-10) passage levels and which was found to be stable during propagation as determined after 1, 2, 5 and 9 weeks
  • Figure 14 - is a bar graph representing analysis of the percentage of hESC cultured on two independent human embryonic fibroblast cell lines (HEFGl and
  • Figures 15A-15I are immunofluorescent images of EBs-derived differentiated cells expressing ⁇ -tubulin (Figs. 15A 5 15D and 15G) 5 AFP (Figs. 15B 5 15E and 15H) 5 desmin (Figs. 15C and 151), or muscle-actin (m- actin, Fig. 15F) when cultured on cord-derived feeders (Figs. 15A-15C); on human embryonic fibroblasts (Figs. 15D-15F); and on foreskin derived feeders (Figs. 15G-15I).
  • Figure 16 - is a bar graph showing the effect of bFGF at the indicated concentration on the number of cells that were harvested per flask at the time of the culture split as shown.
  • Figure 17A-17D - are phase contrast images of cord-derived feeders, showing the effect of bFGF on their morphology after prolonged propagation in the presence of serum without bFGF-supplementation (Fig. 17A) or with the two indicated concentrations of bFGF supplementations (Fig. 17B and Fig. 17C), FACS analysis of the percentage of feeders expressing CD44 and that are immunoreactive with anti-fibroblast antibody (Anti-fib ab) is also shown (Fig. 17D). Analysis was performed at passage 10 in the presence of serum, and at passage 17 when the medium was supplemented with 5ng/ml and 10 ng/ml of bFGF.
  • Figure 18A-18F - are phase contrast images (Fig. 18A-18C) and immunofluorescent images (Fig. 18D-18F) of hESC colonies cultured on cord- derived fibroblasts that were propagated for 17 passages in the presence (Figs. 18B, 18C, 18E and 18F) or absence (Figs. 18A and 18D) of bFGF.
  • the cord-derived fibroblasts supported undifferentiated proliferation of the hESCs as determined by the expression of alkaline phosphatase by the hESC (Fig. 18D-18F) and the expression of stem cell markers by a high percentage of the hESCs (FACS analysis, Fig. 18G).
  • Figure 19 - is a phase contrast micrograph of hESC cultured on HEF feeder layer in Cellgro medium supplemented with 1% TCH showing that hESC retain the morphology of undifferentiated pluripotent stem cells when TCH is used as the serum replacement supplement.
  • Figure 20 - is a bar graph showing the percentage of SSEA-4 expressing on hESC, when cultured on a foreskin or HEF feeder layers, being similar when the KO DMEM was supplemented with KO SR 5 2% TCH, or 2% Nutridoma and showing that Nutridoma-CS is as effective as TCH in supporting undifferentiated propagation of hESC.
  • Figure 21 - is a phase contrast micrograph of hESC colonies cultured within NBN2 showing that hESCS retain the morphology of undifferentiated cells when colonies are cultivated on human feeders in NBN2.
  • Figure 22A-22D - are dark field micrographs of small transparent clusters of cells that develop 7 days after transfer of undifferentiated hESCs into suspension culture within NBN2 medium (Fig. 22A) and after 3 weeks in suspension culture within NBN2, indirect immunofluorescent analysis showed that the majority of hESCs express SSEA4 (Fig. 22B) and Oct4 (Fig. 22D). Nuclei of cells in D are counterstained with DAPI in (Fig. 22C).
  • Figure 23A-23B - are bar graphs showing the percentage of SSEA-4+ cells (Fig. 23A) and total number of cell/well (Fig. 23B) as analyzed after 3 weeks suspension culture of equal initial numbers of hESC in NBN2 medium + FGF2 supplemented with various combinations of ECM components and factors.
  • Figure 24A-24D - are dark field micrographs of EBs that were cultured for 4 weeks in the presence and absence of NA (the culture medium included 10% FCS). EBs with typical cystic structures (cystic EBs) developed in the absence of NA (Figs. 24A and 24B), while in the presence of NA, cystic formation was not observed and the EBs were comprised of tightly packed cells (Figs. 24C and 24D).
  • Figures 25A-25P - are dark field micrographs of EBs that were cultured for 2-5 weeks in chemically-defined medium (NBN2) in the presence or absence of NA and retinoic acid as indicated. EBs with typical cystic structures (cystic EBs) developed in the absence of NA (Figs.
  • Figure 26 - is a RT-PCR analysis demonstrating that the expression of the endodermal marker ⁇ -fetoprotein is suppressed within EBs that differentiated in the presence of NA in comparison to control EBs that were cultured in the absence of NA. After 4 weeks of differentiation, the effect of NA was more prominent in comparison to the effect after 2 weeks.
  • Figures 27A-27D - are immunocytochemical studies demonstrating suppressed expression of ⁇ -fetoprotein (AFP) and cytokeratin-8 (CK) in EBs that differentiated in the presence of NA. Following 4 weeks of differentiation in the presence of NA, only a few cells in sections of EBs were immunoreactive with anti- ⁇ -fetoprotein (Fig. 27A) and cytokeratin-8 (Fig. 27B). Cells that expressed ⁇ -fetoprotein (Fig. 27C) and cytokeratin-8 (Fig. 27D) were abundant within sections of control EBs that differentiated in the absence of NA.
  • Figure 28A-28D - are dark field micrographs of EBs differentiating in the presence of NA showing that the percentage of EBs that included clusters of differentiated cells expressing melanin increased with time (Figs. 28 A, 28B, 28C and 28D representing results after 2, 4, 6 and 12, respectively).
  • Figure 29 - is a real time PCR analysis of EBs differentiated for 6 weeks in the presence or absence of NA, demonstrating the induction of expression of RPE markers by NA.
  • Figure 31 - is RT-PCR analysis showing the expression of chordin-like 1 by cells within EBs that were developed in the presence of NA.
  • Figure 32A-C - are H&E and fluorescent images demonstrating the survival of transplanted hESC-derived RPE cells and their integration within the host RPE layer of cells.
  • An H&E image showing the survival of an intra-vitreal graft, 4 weeks after transplantation into the eye of a mature RCS rat (Fig 32A).
  • the graft includes melanin expressing cells (dark pigmented cells).
  • Indirect immunofluorescent staining demonstrates that the cells within the graft express GFP (white spots), confirming their human identity (Figure 32B).
  • Integration of transplanted hESC-derived RPE cells (pigmented cells marked with arrows) in the albino rat RPE layer is also demonstrated (Figure 32C). Pigmented cells were not observed in the RPE layer of control non transplanted eyes.
  • the invention is described in the following detailed description with reference to cell cultures and culture systems for handling stem cells, preferably human embryonic stem cells. It should be noted that in addition to the cell cultures and culture systems discussed in detailed hereinbelow, also encompassed within the present invention are uses of specific components described with reference to the culture system in the preparation of such culture systems as well as to methods of use of the culture system in handling stem cells cultures and methods of preparing culture cells.
  • a culture system includes one or more culture systems.
  • the term “or” means one or a combination of two or more of the listed choices
  • the term “comprising” is intended to mean that the methods or composition includes the recited elements, but not excluding others.
  • “consisting essentially of” is used to define methods and systems that include the recited elements but exclude other elements that may have an essential significance on the functionality of the culture systems of the inventions.
  • a culture system consisting essentially of a basic medium, medium supplements and feeder cells will not include or include only insignificant amounts (amounts that will have an insignificant effect on the propagation and differentiation of cells in the culture system) of other substances that have an effect on cells in a culture.
  • compositions consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method. "Consisting of shall mean excluding more than trace elements of other elements. Embodiments defined by each of these transition terms are within the scope of this invention.
  • stem cells refers to cells which are capable of differentiating into other cell types having a particular, specialized function (i.e., “fully differentiated” cells) or self renewing and remaining in an undifferentiated pluripotential state as detailed below.
  • the term "cell” refers to a single cell as well as to a population of (i.e. more than one) cells.
  • the population may be a pure population comprising one cell type. Alternatively, the population may comprise more than one cell type.
  • the term "cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g. with an immortal phen ⁇ type), primary cell cultures, finite cell lines (e.g., non- transformed cells), and any other cell population maintained in vitro.
  • the te ⁇ n "primary cell” is a cell which is directly obtained from a tissue, or organ of an animal, including a human, in the absence of culture. Typically, though not necessarily, a primary cell is capable of undergoing ten or fewer passages in vitro before senescence and/or cessation of proliferation. In contrast, a "cultured cell” is a cell which has been maintained and/or propagated in vitro for ten or more passages
  • Non-limiting examples of stem cells are hematopoietic stem cells obtained from bone marrow tissue of an individual at any age or from cord blood of a newborn individual, embryonic stem (ES) cells obtained from the embryonic tissue formed after gestation (e.g., blastocyst), or embryonic germ (EG) cells obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation.
  • ES embryonic stem
  • EG embryonic germ
  • Stem cells can be obtained using well-known cell-culture methods.
  • hESC can be isolated from human blastocysts.
  • Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos.
  • IVF in vitro fertilized
  • a single cell human embryo can be expanded to the blastocyst stage.
  • the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting.
  • the ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth.
  • the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re- plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 1-2 weeks.
  • ES cells For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133, 1998; Proc. Natl. Acad. Sci.
  • stem cells can be also be used in accordance with the invention.
  • Human ES cells can be purchased from the NIH human embryonic stem cells registry.
  • Non-limiting examples of commercially available embryonic stem cell lines are BGOl, BG02, BG03, BG04, CY12, CY30, CY92, CYlO, TE03 and TE32.
  • hESC hESC derived cells
  • diseases presently expected to be treatable by therapeutic transplantation of hESC or hESC derived cells include Parkinson's disease, cardiac infarcts, juvenile-onset diabetes mellitus, and leukemia [Gearhart J. Science 282: 1061-1062, 1998; Rossant and Nagy, Nature Biotech. Yl: 23-24, 1999].
  • feeder cells can secrete factors needed for stem cell self-renewal and proliferation, while at the same time, inhibit their differentiation.
  • Commonly used feeder cells includes a primary mouse embryonic fibroblast (PMEF), a mouse embryonic fibroblast (MEF), a murine fetal fibroblast (MFF), a human embryonic fibroblast (HEF), a human fetal muscle cell (HFM), a human fetal skin cell (HFS), a human adult skin cell, a human foreskin fibroblast '(HFF), a human adult fallopian tubal epithelial cell (HAFT) and a human marrow stromal cells (hMSCs).
  • PMEF primary mouse embryonic fibroblast
  • MFF murine fetal fibroblast
  • HEF human embryonic fibroblast
  • HMF human fetal muscle cell
  • HFS human fetal skin cell
  • HFF human adult skin cell
  • HFF human foreskin fibroblast '(HFF)
  • HAFT human
  • undifferentiated pluripotential hES cells As used herein, the term "undifferentiated pluripotential hES cells" or
  • hESC refers to human precursor cells that have the ability to form any adult cell. Such cells are true cell lines in that they (i) are capable of indefinite proliferation in vitro in an undifferentiated state; and (ii) are capable of differentiation to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture. Human ES cells are derived from fertilized embryos that are less than one week old.
  • Pluripotent SC present at their surface or express biological markers which are used to identify pluripotent SC as well as to verify that the cells in the culture are maintained in an undifferentiated state [Thomson JA et al. Embryonic Stem Cell Lines Derived from Human Blastocysts Science 282(5391):1145 - 1147 (1998)].
  • a " non-limiting list of such cell markers comprise stage-specific embryonic antigen such as SSEA-3, SSEA-4; antibodies to specific extracellular matrix molecule which are synthesized by undifferentiated pluripotent SC, such as TRA-1-60, TRA- 1-81 and GCTM-2; elevated expression of alkaline phosphatase which is associated with undifferentiated pluripotent SC; transcription factors unique to pluripotent SC and which are essential for establishment and maintenance of undifferentiated SC, such as , OCT-4 and Genesis [Carpenter, m.k., Rosier, E., Rao M.S., Characterization and Differentiation of Human Embryonic Stem Cells. Cloning and Stem Cells 5, 79- 88, 2003].
  • stage-specific embryonic antigen such as SSEA-3, SSEA-4
  • antibodies to specific extracellular matrix molecule which are synthesized by undifferentiated pluripotent SC, such as TRA-1-60, TRA- 1-81 and GCTM-2
  • Non-species specific feeder cell technology reduces the value of stem cell cultures due to the foreign nature of the source of the feeder cell.
  • non-species specific feeder cells contain both foreign cells and foreign growth factors.
  • the use of non-species specific feeder cells in combination with different but desirable cultured cells cannot provide the optimum growth conditions as species specific derived feeder cells or conditioned media.
  • the issue of cross-species contamination is particularly relevant to agricultural animals, endangered species, laboratory animals, non-human primate cells, and hESC.
  • hESC are contaminated by foreign molecules when cultured with mouse-derived feeders (Martin, M.J., et al., Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med. 2005; 11: 228-32). Contamination of hESC by mouse derived molecules/pathogens may interfere with their exploitation as a model for basic research and raises concerns as to their use in transplantation therapy. Still further, non-human feeder cell technology reduces the value of human derived SC cultures, as, for example, such non-human feeder cells contain both non-human cells and non-human growth factors. Also, it is believe that the use of non-human feeder cells in combination with human cultured cells cannot provide the optimum growth conditions as human derived feeder cells.
  • the present invention provides, in accordance with a first of its aspects, a cell culture derived from human umbilical cord tissue, preferably excluding hematopoietic tissue, and being capable of maintaining SC in an undifferentiated state when co-cultured therewith.
  • These feeder cells are obtained from culturing, preferably in an animal free culture system, of cells taken from umbilical cord tissue under conditions which allow the cells to propagate/expand and isolating the thereby propagated cells.
  • the cells in the culture are essentially fibroblast cells and are preferably used as feeders in stem cell culture systems.
  • the cell cultures in accordance with the invention may be a fresh cell culture, cryopresrved culture as well as cryopreserved and thawed cells.
  • human embryonic cord derived cells denote, in accordance with one embodiment of the invention, fibroblast cells originating from embryonic cord tissue, which under suitable condition propagate into a fibroblast cell line.
  • feeder cells which is known interchangeably with “feeders” denotes any type of cells which may be used as a substratum for other cells attachment and growth in a culture system.
  • Feeder cells are typically used to allow growth and survival of single undifferentiated stem cells.
  • the Feeder cells provide conditions that maintain cell proliferation, inhibit cell differentiation and preserve pluripotency.
  • the feeder cells are cells that secrete factors needed for stem cell proliferation, while inhibit their differentiation. Methods of preparing feeder cells are well known in the art (see, for example, U.S. patent Pub. No. 20030143736).
  • the feeder cells may be fibroblasts or other types of cells, and the cells are inactivated by large- dose radiation before use, such as ⁇ -ray, or by drugs, such as mitomycin C. After the inactivation process, the surviving cells lost the capability to proliferate, but retained their physiological functions, such as metabolism and synthesis of growth factors.
  • the feeder cells are derived (expanded) from umbilical cord tissue.
  • Umbilical cord tissue may be obtained in the course of vaginal delivery.
  • a major advantage of using umbilical cord tissue is that it may be obtained during elective cesarean section in a sterile environment of an operating theater.
  • the umbilical cord is obtained from the sterile environment of the amniotic sac and has not been exposed to any external contagious agents prior to donation. The sterile nature of umbilical cord donation allows the derivation of feeders from the umbilical cord tissue without the use of antibiotics or anti-fungal drugs.
  • Avoiding the use of anti-bacterial and anti-fungal drugs is an advantage since these drugs may interfere with the growth of cells in culture, alter the results of basic science studies and most importantly may induce allergic reactions in, recipients of cells that were cultured in the presence of these drugs.
  • Derivation of feeders from other human primary tissues such as foreskin or aborted fetuses are done under significant less sterile conditions.
  • the foreskin is exposed to bacteria that colonize the genital area and it may be disinfected but not sterilized.
  • Aborted fetuses are also exposed to potential contamination by vaginal and genital flora during dilatation and curettage.
  • umbilical cord as opposed to foreskin or human fetal tissues is that a significant volume of blood may be sampled from the umbilical cord, tested for contagious agents and archived. This is not possible with foreskin tissues donated by newborn babies or with aborted fetuses. Lastly, umbilical cord is routinely discarded and its donation is not associated with emotional or moral constrains, while donation of fetal tissues raises ethical concerns and is not morally accepted by many.
  • umbilical cord derived feeder cells comprising isolating umbilical cord cells from umbilical cord tissue and culturing said umbilical cord cells in a culture medium including serum, thereby preparing said human umbilical cord feeder cells.
  • the umbilical cord cells may be isolated from the umbilical cord tissue by mincing the tissue and affixing the umbilical cord to a wall, such as a wall of a flask, and allowing the cells to incubate undisturbed for a number of weeks until fibroblast cells begin to migrate out of the minced umbilical cord tissue.
  • the umbilical cord tissue may be obtained from healthy pregnant women undergoing elective Cesarean sections at term.
  • a culture system for maintaining stem cells (SC) in an undifferentiated state comprising feeder cells selected from cells obtained from human umbilical cord tissue (excluding cells obtained from umbilical cord blood), human embryonic fibroblast cells (HEF) or a combination of same.
  • the culture system according to this aspect of the invention is term herein the "human derived feeder cell aspect of the invention”.
  • the terms “maintenance” means continued survival of a cell or population of cells, at times, with an increase in numbers of cells.
  • this term refers to a continuous survival of the cells for at least 10 weeks.
  • the culture systems in accordance with the invention are preferably for enabling maintenance of a population of stem cells when cultured on feeder cells, and at time, propagation of same, for a prolonged period of time, the period of time being at least 10 weeks.
  • the feeder cells are essentially fibroblast cells.
  • the term "essentially fibroblast cells” denotes that the feeder cells comprise in its majority fibroblasts, i.e. at least 70 of the cells in the feeder cell population are fibroblast, preferably 85%, a most preferably all the cells, i.e. essentially 100% of the feeder cells are fibroblasts.
  • the feeder cells are provided in a form of a monolayer coated culture dish to which a nutrient medium is added along with the culture cells.
  • a nutrient medium is added along with the culture cells.
  • the terms “monolayer”, “monolayer culture” and “monolayer cell culture” refer to cells that have adhered to a substrate and grow as a layer that is one cell in thickness.
  • Monolayer cells may be grown in any format, including but not limited to flasks, tubes, coverslips (e. g., shell vials), roller bottles, etc.
  • Monolayer cells may also be grown attached to microcarriers, including but not limited to beads. At times, the term monolayer also includes growth of cells as flat colonies.
  • the term "culture system” denotes a combination of elements, such as an extracellular matrix (ECM) and a culture (nutrient) medium which together provide suitable conditions that support SC growth.
  • the conditions are such that SC can proceed through the cell cycle, grow and divide.
  • the conditions are such which enable growth of human stem cells, preferably, hESC.
  • the culture system provides conditions that permit the embryonic stem cells to stably proliferate in the culture system for at least 10 weeks.
  • the nutrient medium may contain any of the following appropriate combinations: a basic medium (a cell culture medium usually comprising a defined base solution, which includes salts, sugars and amino acids) as well as serum or serum replacement, and other exogenously added factors.
  • the culture system also comprises the feeder cells.
  • the feeder cells may be substituted with components derived from feeder cells or other known and acceptable substitutes thereof, e.g. when referring to other culture systems disclosed herein.
  • the culture system is employed for maintaining hESC in an undifferentiated pluripotential state, as evidenced in the following non-limiting examples by the expression of proteins such as SSEA-4, TRA- 1-60, OCT-4, APase, but not SSEA-I.
  • proteins such as SSEA-4, TRA- 1-60, OCT-4, APase, but not SSEA-I.
  • Methods of preparing culture systems for culturing hESC are well known in the art [see, for example, Reubinoff Be. et. al., Nat. Biotechnol. 18:399-404, 2002; Richards, M. et al, Nat. Biotechnol. 20:933-936, 2002].
  • a hESC medium may typically contain 80% Dulbecco's Modified Eagles
  • DMEM Fetal Calf Serum
  • 1% L-Glutamine 0.5% penicillin/streptomycin
  • 1% non-essential amino acids 1% Insulin-Transferrin- Selenium G supplement and 1 mM ⁇ -mercaptoethanol.
  • the cultures rely on human feeder layers supplemented with human serum or serum replacement suitable for the growth of human stem cells.
  • the feeder cells may be any suitable cells from human source as known in the art or the isolated umbilical cord derived feeder cells of the invention; the stem cells medium DMEM (used as the basic media) may be replaced with KO DMEM (Gibco, or equivalent), X- Vivo 10 (Biowhittaker, Maryland, or equivalent) or Cellgro Stem Cell Growth Medium (CellGenix, Freiburg, Germany, or equivalent); the FCS may be replaced with humanized serum replacement substitute, such as TCH (Protide Pharmaceuticals, St.
  • the method comprises incubating (co-culturing) said cells with a culture system comprising feeder cells selected from human umbilical cord tissue derived cells, human embryonic fibroblast cells (HEF) or a combination of same.
  • a culture system comprising feeder cells selected from human umbilical cord tissue derived cells, human embryonic fibroblast cells (HEF) or a combination of same.
  • the stem cells are incubated in a culture system where the feeder cells are preferably provided as a layer of cells, preferably a mono-layer, formed on a base of culture dish.
  • the culture system is then provided with a growth environment, typically, an environment in which cells of interest will proliferate in vitro. Temperatures of 37 0 C and 5% CO 2 in air are generally adopted.
  • the invention also provides a culture system for inhibiting or preventing differentiation of stem cells towards extraembryonic lineages (to extraembryonic cells).
  • the culture system in accordance with this aspect of the invention comprise NA (NA) or a derivative of NA having an inhibitory effect on differentiation of stem cells towards extraembryonic lineages (to extraembryonic cells) similar to that of NA.
  • NA NA
  • a derivative of NA having an inhibitory effect on differentiation of stem cells towards extraembryonic lineages (to extraembryonic cells) similar to that of NA.
  • This aspect of the invention is referred to herein as the "nicotinamide aspect of the invention”.
  • NA is a form of Vitamin B3 that may preserve and improve beta cell function.
  • NA is essential for growth and conversion of foods to energy and it has been used in diabetes treatment and prevention. It has now been found that NA is capable of inhibiting, preferably, preventing differentiation of embryonic stem cells towards extraembryonic lineages (to extraembryonic cells).
  • nicotinamide denotes a compound which is a chemical modification of the natural NA.
  • the chemical modification may include substitution on the pyridine ring of the basic NA structure (via the carbon or nitrogen member of the ring), via the nitrogen or the oxygen atoms of the amide moiety, as well as deletion or replacement of a group, e.g. to form a thiobenzamide analog of NA, all of which being as appreciated by those versed in organic chemistry.
  • the derivative in the context of the invention also includes the nucleoside derivative of NA (e.g. nicotinamide adenine).
  • NA derivatives are described, some also in connection with an inhibitory activity of the PDE4 enzyme [WO03068233; WO02060875; GB2327675A], or as VEGF-receptor tyrosine kinase inhibitors [WO01/55114].
  • PDE4 enzyme WO03068233; WO02060875; GB2327675A
  • VEGF-receptor tyrosine kinase inhibitors WO01/55114.
  • the NA derivatives in the context of the invention are compound determined to have an inhibitory effect, preferably preventative effect, on differentiation of stem cells to extraembryonic lineages (extraembryonic cells), similar to that of NA.
  • NA may be the result of inhibition of poly (ADP-ribose) polymerase (PARP). Therefore the effect of NA may be also achieved by treating the cells with other PARP inhibitors such as 3-aminobenzmide, PJ-34 or 1, 5- dihydroxyisoquinoline. These other PARP inhibitors are also included in the context of the term "modification of NA”. Yet further, the effect of NA may also be attributed to the inhibition of SIRT protein deacetylase. Therefore its effect may be also obtained by other SIRT inhibitors such as splitomicin and sirtinol, which are thus, also included in the context of the term term "modification of NA".
  • SIRT inhibitors such as splitomicin and sirtinol
  • the stem cells may be as described above, i.e. they may be stem cells from any source, but are preferably human stem cells, further preferably, human embryonic stem cells.
  • inhibition of extraembryonic differentiation used synonymy with the term “prevention of extraembryonic differentiation” denotes the maintainance as well as the expansion of embryonic stem cell in a cell culture and that the resulting cell culture is essentially free of extraembryonic cells or membranes.
  • the term "essentially free” is used to exclude extraembryonic cells that may have an essential significance on the functionality of the stem or somatic cells in the culture or that the amount of the extraembryonic cells in the cell culture is insignificant (an amount that will have an insignificant effect on the propagation and differentiation of cells in the culture system). It is well appreciated that if extraembryonic differentiation is essentially eliminated, a key challenge is to further direct differentiation into a specific somatic lineage and into a specific type of cell. It has now been found that supplementation of a culture medium with NA can prevent the default differentiation of hESCs towards extraembryonic lineages. It may also direct the differentiation towards specific somatic lineage such as but not limited to neural differentiation. The examples provided herein show differentiation to neural precursor cells.
  • NA is similar ( Figure IA in Vaca P .et al.), insulin content is increased in cells that differentiate in the presence of NA. Nevertheless it is unclear whether the increased insulin content was not related to increased uptake of insulin from the medium.
  • RPE retinal pigmented epithelial
  • RPE cells may serve as a vector to carry and express genes in the retina after transplantation.
  • Other applications may be the use of hESC-derived RPE cells as an in vitro model for the development of new drugs to promote RPE survival and function.
  • hESC-derived RPE cells may serve for high throughput screening for compounds that are toxic, trophic, induce differentiation proliferation and survival of RPE cells. They may be used to uncover mechanisms, new genes, soluble or membrane-bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells.
  • the culture system in the NA aspect of the invention comprises standard elements of culture media, as defined above combined with NA.
  • concentration of NA in the medium may vary, however, will preferably be in a concentration range between about ImM to about 2OmM, more preferably at a concentration of about 1OmM.
  • a method for inhibiting or preventing differentiation of stem cells towards extraembryonic lineages comprises incubating said stem cells in a culture system comprising NA or a derivative of
  • NA based culture systems was also effective for increasing the survival of SC in the culture system. According to one embodiment, cells survived in the culture for at least 12 weeks.
  • the NA based culture system of the invention was effective to induces an increase in number of cells within embryoid bodies (EB) cultured therein.
  • the stem cells in accordance with the NA aspect of the invention are preferably grown as free floating clusters in a suspension.
  • the terms "suspension” and “suspension culture” refer to cells that survive and proliferate without being attached to a substrate.
  • a further aspect of the invention concerns the use of serum (e.g., fetal bovine serum (FBS)), in SC cultures. It has already been established that serum is a major source of undefined differentiation factors and thus tends to promote ES cell differentiation. Other problems are also " associated with serum.
  • FBS fetal bovine serum
  • the invention also provide a humanized culture system for maintenance of stem cells (SC) in an undifferentiated state, the humanized culture system comprising animal free stem cell basic medium and a humanized serum replacement substitute.
  • SC stem cells
  • This aspect of the invention is referred to as the "humanized serum free culture system of the invention”.
  • the humanized culture system comprises a serum free basic medium as known to those versed in the art of stem cells (i.e. a medium which is free of animal origin and is suitable for growth of stem cells), selected from Cellgro Stem Cell Growth Medium, KO DMEM,
  • the humanized culture system comprises a serum replacement substituent selected from TCHTM, Nutridoma-CS or combination of same.
  • the culture system further comprises N2 supplement [GIBCO® Cell Culture] or a modified N2 supplement, the modification rendering the medium supplement suitable for use with stem cells.
  • N2 supplement [GIBCO® Cell Culture] or a modified N2 supplement, the modification rendering the medium supplement suitable for use with stem cells.
  • the standard and commercially available N2 supplement comprises insulin, transferrin, progesterone, putrascine, selenite.
  • N2 supplement as published by StemCell Technologies Inc (Product Information Sheet, revised on December 2002) includes 2.5mg/mL rh insulin, lOmg/mL human transferring (which may be iron-poor or iron-saturated), 0.52 ⁇ g/mL sodium selenite, 1.61mg/mL putrascine, 0.63 ⁇ g/mL progesterone, all in phosphate buffered saline. Nonetheless, modifications of the standard N2 supplement for stem cells maintenance are readily envisaged by those versed in the art.
  • TCHTM is a completely biochemically defined serum replacement developed primarily for human cells and production of cell-secreted proteins.
  • TCHTM may be purchased from Protide Pharmaceuticals (MN, USA) as well as from BM Biomedicals (CA, USA).
  • Nutridoma-CS is also a completely biochemically defined serum free medium supplement composed of albumin, insulin, transferrin, cytokines a cholesterol source and other defined organic and inorganic compounds.
  • the use of humanized SR as suggested herein in combination with serum free basic mediums thus enables the providence of an animal free and humanized culture system for maintenance as well as expansion of undifferentiated SC, preferably human SC 5 more preferably, bJESC.
  • the culture system may comprise, in addition to the humanized SR, human derived feeder cells such as that disclosed above (as well as those known in the art),
  • the amount of the humanized SR may vary and will depend on other elements forming part of the culture system. Those versed in the art will know how to manipulate the concentrations of SR in the culture system to facilitate maintenance of the stem cells cultured therewith. According to one embodiment, 2% TCH may be used. However, higher or lower concentrations of TCH may be used.
  • humanized SR aspect of the invention there is also provided the use of humanized serum replacement substitute selected from TCHTM, Nutridoma-CS or combination of same for the preparation of a culture system for maintaining stem cells, preferably human embryonic stem cells, in an undifferentiated state.
  • stem cells preferably human embryonic stem cells
  • the cells are co-cultured with feeder cells, preferably human derived feeder cells.
  • the invention provides a further culture system for maintaining a stem cells, preferably human, more preferably, hESC, in an undifferentiated state, the culture system comprising NeurobasalTM medium.
  • the Neurobasal is supplemented with N2 supplement or a modification of N2 supplement (as defined above) for a humanized flat culture system of hESC on feeders (see for example Fig. 21) as well as for maintenance of stem cells in suspensions (see for example Fig. 22).
  • NeurobasalTM is known in the art of cell cultures [Brewer GJ. Serum-free B27/Neurobasal medium supports differential growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, J Neurosci Res.
  • NeurobasalTM based culture aspect of the invention As indicated above, and in accordance with one embodiment of this aspect of the invention the culture system is supplemented with N2 supplement, a chemically-defined additive for NeurobasalTM Media.
  • ECM extra cellular matrix
  • Structural proteins collagen and elastin.
  • Specialized proteins e.g. fibrillin, fibronectin, and laminin.
  • Proteoglycans conjugates of a protein core and glycosaminoglycans
  • Additional elements may include growth factors, for example, without being limited thereto, FGF (e.g. FGF2) as well as others known in the art (see also Fig. 23A and 23B).
  • FGF e.g. FGF2
  • additional elements which may be added include noggin and activin A, as familiar to those versed in the art of stem cells.
  • a method for maintaining SC preferably human, more preferably, hESC, in an undifferentiated state, the method comprising incubating the SC with a culture system comprising NeurobasalTM in a growth environment, in which cells of interest will proliferate in vitro, as detailed herein.
  • Human ESC (HESl and HES2 cell lines) were cultured on the human feeder layers in KO medium (KOM) consisting of 85% KO-DMEM, 15% KOSR, 1 mM glutamine, 0.1 mM ⁇ -mercaptoethanol, 1% nonessential amino acids, 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, (Gibco, Gaithersburg, MD) and 4 ng/ml bFGF (R&D Systems, Inc., Minneapolis, MN).
  • hES cells were weekly passaged by dissociation into nearly single cell suspension with Ca/Mg ⁇ -free PBS supplemented with 0.05% EDTA (Biological Industries, Beit Haemek, Israel) and plated onto fresh feeder layer.
  • Human ES cells were removed from the feeders by treatment with dispase (10mg/ml; Gibco), and/or type IV collagenase (lmg/ml; Gibco). The clusters of undifferentiated cells that were obtained were further triturated into smaller clumps within PBS.
  • clumps were cultured for various periods in suspension within bacteriological dishes precoated with 0.1% low melting temperature agarose in DMEM (Gibco), supplemented with 10-20% FCS (Biological Industries,Beit Haemek ), ImM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, 1% non-essential amino acid stock, 50 units/ml penicillin, 50 ⁇ g/ml streptomycin (all from Gibco Invitrogen Corporation products, USA) in the presence or absence of nicotinamide 1OmM (Sigma).
  • FCS Biological Industries,Beit Haemek
  • ImM L-glutamine ImM L-glutamine
  • 0.1 mM ⁇ -mercaptoethanol 1% non-essential amino acid stock
  • 50 units/ml penicillin 50 ⁇ g/ml streptomycin (all from Gibco Invitrogen Corporation products, USA) in the presence or absence of nicotinamide 1OmM (Sigma).
  • FCS
  • EBs were dissociated into smaller clusters mechanically with/without the aid of trypsin (0.025%, 3mM EDTA in PBS) digestion.
  • the small clusters of cells were plated on poly-D-lysine (30-70 kDa, 10 ⁇ g/ml; Sigma, St. Louis, MO) and laminin-coated (4 ⁇ g/ml; Sigma) glass coverslips and cultured for additional 3-5 weeks in the culture medium used for suspension culture of EBs or neural spheres. Differentiated cells within the outgrowth were fixed with 4% paraformaldehyde for 20 minutes at room temperature.
  • PCR was carried out using standard protocols with Taq DNA Polymerase (Promega). Amplification conditions were as follows: denaturation at 94°C for 60 seconds, annealing at 55-6O 0 C for 60 seconds, and extension at 72 0 C for 60 seconds. The number of cycles was 35 Primer sequences and lengths of amplified products were:
  • the length of amplification product was 338bp.
  • Clusters of pigmented cells were mechanically isolated by scalpel blades from EBs that were differentiating in the presence of NA for 6-8 weeks.
  • the clumps were dissociated into smaller clusters of cells by digestion with Papain (Papain Dissociation System; Worthington Biochemical Corporation, Lakewood, New Jersy) at 37 0 C for 30 minutes followed by trituration.
  • Male (body weight 230-25Og) outbred RCS and albino rats were used for intraocular transplantation. All animal experiments were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the institutional committee for animal research.
  • Ketamine HCl Ketalar, Parke Davis, UK, 100mg/kg
  • Xylazine 2.0mg/kg
  • Local anaesthetic drops Benoxinate HCl 0.4%, Fischer Pharmaceuticals, Israel
  • the pupils were dilated with Tropicamide 0.5% (Mydramide, Fisher Pharmaceuticals, Israel) and Phenylephrine HCl 2.5% (Fisher Pharmaceuticals, Israel).
  • specimens were de-parafmized in xylene and dehydrated in graded alcohols, rinsed with phosphate-buffered saline (PBS, pH 7.4), and incubated with 10 mM citrate buffer (pH 6.0) at 110 0 C for 4 minutes. After washing with PBS, specimens were blocked for 1 hour at room temperature with PBS solution containing 1% bovine serum albumin, 0.1% triton-xlOO, and 3% normal goat serum. Subsequently, sections were incubated for 24 hours at 4 0 C in a humidified chamber with anti-green fluorescent protein (anti-GFP; Santa Cruz Biotechnology; rabbit polyclonal, 1 :100).
  • anti-GFP Santa Cruz Biotechnology
  • Human feeders were obtained via Informed Consent from aborted fetuses (10-12 weeks of gestation), umbilical cords, and newborn foreskin.
  • Term umbilical cord tissue was minced as above, and the small pieces of tissue were plated in the fibroblast culture medium, as described above. To promote the adherence of the tissue pieces to the culture dish, flasks were incubated upright overnight. Cells emanating from the tissue pieces were propagated as above.
  • Foreskin tissue was obtained from 7 day to 6 months circumscribed newborns and babies. Circumcision was performed in the operating room, and the medium described above was used for the development and culture of the foreskin feeders. The circular foreskin was cut and spread on the tissue culture dish. The epidermis was scraped with scalpel blade followed by washing with the culture medium. This was repeated 5 times and small foreskin tissue pieces were plated in the culture medium within a flask. The flask was incubated upright to enhance the adherence of tissue pieces to the dish and promote outgrowth of fibroblasts.
  • AU fibroblasts were cryopreserved in human serum (Cambrex, Maryland, Maryland) supplemented with 10% Cryosure-DMSO (Wak-Chemie, Germany). Slow-cooling and rapid-thawing standard methods were used.
  • fibroblasts were plated and cultured on cover slides. They were fixed with 4% paraformaldehyde for 20 minutes at room temperature, and then permeabilized with 0.2% Triton XlOO (Sigma) in PBS for 5 minutes. The cells were incubated with anti human vimentin (mouse monoclonal IgG2a, K Dako, 1:10). Primary antibody localization was performed by using fluorescein isothiocyanate (FITC) - conjugated goat anti-mouse immunoglobulins (Dako, 1:50).
  • FITC fluorescein isothiocyanate
  • FACS analysis of marker expression by the feeders was performed by using a FACS Calibur system (Becton-Dickinson, San Jose, CA). Propidium Iodide was added (final concentration of 4 ⁇ g/ml) for better gating of viable cells.
  • the feeders were disaggregated using TrypLE Select solution (Gibco,Gaithersburg, MD). The cells were " then washed with FACS media consisting of PBS supplemented with 1% BSA and 0.05% sodium azide. The single cell suspension was stained with
  • Anti-human fibroblasts antibodies (mouse monoclonal IgG2a, Acris,
  • Control feeders were stained with an isotype control antibody.
  • Primary anti-human fibroblasts antibodies were detected with a FITC-labeled goat anti-mouse Ig (1 :100, Dako).
  • Human embryonic fibroblasts derived from umbilical cord as described herein or foreskin fibroblasts were used as feeders.
  • the feeders were plated in tissue culture dishes precoated with 1 ⁇ g/cm 2 human Fibronectin (BD Biosciences, Bedford, MA) or 100 ⁇ g /ml recombinant Gelatin (FibroGen, SF). Mitotic inactivation was carried out by incubating the feeders 2.5 hours with Mitomycin-C (Kyowa, Tokyo).
  • KO DMEM Gibco
  • X-Vivo 10 Biowhittaker, Maryland
  • Cellgro Stem Cell Growth Medium CellGenix Freiburg, Germany
  • TCH 1- 2%; Protide Pharmaceuticals, St. Paul, MN
  • Nutridoma-CS 2%; Roche, Germany
  • the media were supplemented with 2mM glutamine (Hyclone, Utah) and non-essential amino acids (NEAA, 1%; Hy clone Utah).
  • HESC were split weekly with Ca/Mg ⁇ -free PBS supplemented with 0.05% EDTA.
  • hESC were disaggregated with Ca/Mg ++ -free PBS supplemented with 0.05% EDTA, spun down, and re-suspended in Cellgro medium, supplemented with 2% TCH, and 10% Cryosure-DMSO (Wak-Chemie, Germany). Conventional slow-rate cooling and rapid-thawing methods were used.
  • FACS analysis of marker expression was performed on hESC after disaggregation using Ca/Mg ⁇ -free PBS supplemented with 0.05% EDTA. The cells were then washed with FACS media consisting of PBS supplemented with 1% BSA and 0.05% sodium azide. The single cell suspension was stained with anti-SSEA4 (1:100, mouse monoclonal IgG3 developmental Studies Hybridoma Bank (DHSB) 5 Iowa City, IA), and anti-Tra-1-60 (1:20, monoclonal mouse IgM, gift from Prof.
  • DHSB mouse monoclonal IgG3 developmental Studies Hybridoma Bank
  • HESC colonies were dissociated with 0.05% of EDTA for 7-10 min at 37°, or mechanically with the aid of collagenase IV (lmg/ml, 120 min at 37°). Cell dissociation was promoted by gentle blowing of the medium with a 1 ml pipette tip towards the hESC colonies.
  • the cells/cell-clusters were re-suspended in NBN2 medium (Neurobasal, N2 supplement 1:100, glutamine 2mM, 50 units/ml penicillin, 50 ⁇ g/ml streptomycin) supplemented with bFGF 20ng/ml, noggin 250ng/ml, activin 25-50ng/ml, fibronectin and laminin 5ng/ml each, gelatin 0.001%.
  • the suspension may be strained though 30-50micron mesh to remove big clumps and transferred into tissue culture dishes (Costar ® , Corning Inc., Corning, NY, USA) at a density of ⁇ 0.7-1.2 x 10 6 cells/ml.
  • the cells were dissociated with EDTA solution (as above), followed by gentle trituration, and were plated in NBN2 on glass coverslips, pretreated with poly-D-lysine (30- 70 kDa, 10 ⁇ g/ml; Sigma, St. Louis, MO) ⁇ and laminin (4 ⁇ g/ml; Sigma). After one hour's incubation at 37°, the cells were incubated 1 min in propidium iodide solution (PI, 1 ⁇ g/ml in PBS, Sigma) in order to distinguish dead cells from vital ones. The cells were then washed with PBS and fixed with 4% PFA for 20 min.
  • PI propidium iodide solution
  • the fixed cells were incubated for 30 min at room temperature (RT) with the following primary antibodies: anti-SSEA4 (1:200 of the concentrated monoclonal mouse IgM, Developmental Studies Hybridoma Bank (DHSB), Iowa City, IA), anti-Tra-1-60 (1 :20), anti-Tra-1-81 (1:10) (monoclonal mouse IgM, gift from Prof. P. Andrews), anti-GCTM2 (supernatant, monoclonal mouse IgM, gift from Prof. M. Pera).
  • RT room temperature
  • hESC human embryonic fibroblast
  • HEF human embryonic fibroblast
  • umbilical cord derived fibroblast feeders and foreskin- derived fibroblasts.
  • feeder cells were developed and cultured using a clinical grade animal free culture system comprising the following reagents:
  • DMEM HyQ DME (Hyclone, Utah or equivalent)
  • - Human serum Cambrex, Maryland, or equivalent
  • FCS fetal calf serum
  • - Human fibronectin or recombinant gelatin FibroGen, SF 5 or equivalent
  • TrypleSelect Gibco.Gaithersburg, MD, or equivalent
  • a recombinant enzyme replacing animal-derived trypsin for splitting of fibroblasts
  • Cryosure-DMSO (Wak-Chemie, Germany), or equivalent, a GMP- qualified product for fibroblast cryopreservation
  • Mitomycin-C (USP or equivalent) from a GMP-qualified source for fibroblasts mitotic inactivation.
  • fibroblasts cell lines derived from each of the three groups were characterized between passages 5-8.
  • the cells had a typical fibroblast morphology ( Figures IA and 1C).
  • Immunostaining demonstrated that over 70% of the cells in each of the cell lines were immunoreactive with markers of fibroblasts including but not limited to anti-vimentin ( Figures 2A-2F), anti- CD44 and anti-human fibroblasts antibody ( Figure 3).
  • the karyotype of the feeders was normal ( Figure 4A). Fibroblasts doubling rate was between 20-50 hours (Table 1).
  • the DNA had a definitive human STR profile (not shown).
  • hESCs were cultured on the feeder layers for a period of ten weeks and their phenotype as well as developmental potential were characterized.
  • the phenotype of the hESCs was characterized at two time periods. The first time point, between passages 1-5 and the second time point, between passages 6-10.
  • the hESC were cultured in KO DMEM supplemented with 20% KO SR.
  • the cord-derived feeders could be maintained for prolonged periods (17 passages). It has also now been found that proliferation of the fibroblasts could be augmented by supplementation of the medium with FGF2 (Figure 16). The fibroblasts maintained their typical morphology and marker expression following expansion with or without FGF2 supplementation ( Figure 17A-17D). Cord- derived fibroblasts that were propagated in culture for extended periods of time with or without the addition of FGF2 could support undifferented proliferation of hESC ( Figure 18A-18G).
  • the feeder cells from the three types of primary tissues could be successfully cryopreserved and thawed. Above 55% of the cells survived thawing. The morphology and marker expression and growth rate were not altered by cryopreservation. Cryopreserved feeders from the three sources could support undifferentiated proliferation of pluripotent hESC, as detailed above.
  • cryopreservation solution knockout (KO) DMEM, 10% DMSO, 10% KO serum replacement (SR)
  • SR KO serum replacement
  • cell viability and growth rate post-thawing were similar with both cryopreservation solutions.
  • Animal-free culture system for hESC employed in the exemplary embodiments presented here, included the following components:
  • KO DMEM Gibco, or equivalent
  • X- Vivo 10 Biowhittaker, Maryland, or equivalent
  • KO DMEM, X- Vivo 10, or Cellgro Stem Cell Growth Medium were used as the basic media for the propagation of undifferentiated hESC colonies on Human Embryonic Fibroblast (HEF) or foreskin feeders. These basic media were supplemented with TCH, which is an animal-free, defined serum replacement (SR) substitute. TCH replaced KO SR, which contains animal products and which is most commonly used as a supplement to KO medium.
  • TCH is an animal-free, defined serum replacement (SR) substitute.
  • TCH replaced KO SR, which contains animal products and which is most commonly used as a supplement to KO medium.
  • Nutridoma-CS which is animal free, can also serve as a SR substitute for the support of undifferentiated propagation of hESC. Both TCH and Nutridoma-CS were found to be as effective as KO SR in supporting undifferentiated propagation of hESC.
  • hESC were cultured on human feeders (embryonic fibroblasts or foreskin) in KO DMEM supplemented with KO SR, 2 % TCH 5 or 2 % Nutridoma-CS
  • the percentage of cells expressing SSEA-4 was comparable in the three systems. The percentages were 93 % and 79% with KO+ SR on Foreskin and HEF 5 respectively; 97% and 72 % with KO+ TCH, and 93% and 86% with KO+ Nutridoma-CS ( Figure 20).
  • human fibronectin or recombinant gelatin replaced the animal gelatin that is most commonly used as a matrix to pre-coat the tissue culture dishes.
  • recombinant gelatin when hESC were cultured on a foreskin feeder layer in KO DMEM + KO SR on culture plates precoated with gelatin or recombinant gelatin, the percentage of SSEA-4+ hESC was comparable (91% and 94%, respectively).
  • Penicillin/Streptomycin and ⁇ -Mercaptoethanol were omitted from the hESC culture media without affecting the growth rate or the level of background differentiation.
  • NeurobasalTM (NB) medium which was initially developed for the maintenance of neural cells in ambient atmosphere, and is animal-reagent free, is used as the basic medium of the culture system.
  • N2 supplement which includes insulin, transferrin, progesterone, putrascine, selenite, NBN2
  • the hESC are weekly sub-cultured following mechanical or enzymatic (dispase, type IV collagenase, or trypsin) disaggregation or treatment with Ca/Mg ⁇ -free PBS supplemented with EDTA, or the combination of the above.
  • the hESC retained the morphology of undifferentiated cells ( Figure 21), and expression of alkaline phosphatase (AP).
  • the medium may be supplemented with FGF2.
  • hESC To exploit the potential of hESC for high throughput screening, drug discovery, basic research, regenerative medicine, and other potential applications, large numbers of cells are required. The number of hESC that may be obtained with monolayer cultures is limited. Culture of hESC in suspension rather than in a monolayer is required to develop bulk cultures of hESC. Suspension cultures of hESC may allow extensive expansion of the cells with a bioreactor system. It may allow initiation of differentiation processes in suspension of a large number of cells, and the development of novel methodologies to direct differentiation of hESC within suspension cultures.
  • Neurobasal 1 medium Propagation of hESC in suspension is achievable using Neurobasal 1 medium as the basic medium of the culture system.
  • the NeurobasalTM medium may be supplemented with N2 (NBN2). It may be further supplemented with soluble factors including but not limited to FGFs, TGF ⁇ superfamily factors, BMPs antagonists as well extracellular matrix (ECM) components including but not limited to fibronectin, laminin, gelatin to promote proliferation and survival of the cells and to prevent their differentiation.
  • ECM extracellular matrix
  • hESC colonies that are cultivated on human feeders in the KO culture system are dissociated with the aid of type IV collagenase or Ca/Mg ++ -free PBS supplemented with 0.05% EDTA.
  • the cells/cell clusters that are obtained are re-suspended within fresh NBN2 medium, supplemented with FGFs (bFGF 20ng/ml).
  • BMPs antagonists noggin 250ng/ml
  • ECM components laminin 5ng/ml, fibronectin 5ng/ml, gelatin
  • the cells are transferred to suspension at a density of -0.7-1.2 x 10 6 cells/ml. Dead/fragmented cells are gradually removed during medium refreshment. After 3-5 days of suspension culture, small transparent cell- aggregates develop. These transparent cells proliferate as free-floating tiny clusters of 20-50 cells without any morphological signs of differentiation (Figure 22A). Aggregation and overgrowth of the transparent clusters may be prevented by daily trituration through a 1000 ⁇ l pipette tip or by the use of bioreactor.
  • the cells express SSEA4 and OCT4, ( Figures 22B and 22D respectively). When re- plated on human feeders, after 6 weeks of suspension culture, they give rise to colonies with the morphology of undifferentiated hESCs.
  • Nicotinamide may be added to the medium to prevent the differentiation of the cells towards extraembryonic lineages, to promote their survival and to maintain them undifferentiated.
  • Nicotinamide (KA) for the maintenance of undifferentiated hES cells, prevention of extraembryonic differentiation, and for the induction of somatic differentiation.
  • NA The effect of NA on differentiation within EBs was not dependent on the presence of serum and was observed also when EBs differentiated in a chemically-defined medium (NBN2; Figures 25A-25H, i.e. upper two panels).
  • NA also had an effect on the size of the EBs.
  • serum-free medium supplemented with NA 5 the size of the Ebs after two weeks of differentiation was significantly higher (1.7 times; maximal diameter of EBs) in comparison to the size of EBs that were cultured under the same conditions in the absence of NA (Figures 25E-25F, i.e. left pictures of upper two panels).
  • the increased size of the EBs in the presence of NA reflected an increase in the number of cells within the EBs.
  • NA can be used to increase the number of differentiated cells that are obtained from hESC.
  • Prolonged culture of EBs is important for the completion of complex differentiation processes that require long time periods, and may promote the maturation of differentiated cells.
  • Retinoic acid is known to induce extraembryonic differentiation of human pluripotent stem cells (Roach, S., Schmid, W., Pera, MF. Hepatocytic transcription factor expression in human embryonal carcinoma and yolk sac carcinoma cell lines: expression of HNF-3 alpha in models of early endodermal cell differentiation. Eep Cell Res 215, 189-98 (1994).
  • hESC-derived EBs were cultured in the presence of RA there was extensive cystic formation. NA could block the effect of RA and when EBs were cultured in the presence of RA and NA the development of cystic structures was infrequent ( Figures 25I-25P, i.e. lower two panels).
  • the number of differentiated cells that were obtained in the presence of RA was low. This effect of RA was also blocked when the medium was supplemented with NA ( Figures 25I-25P, lower two panels).
  • RPE cells which lie immediately underneath the photoreceptors and form part of the retina-blood barrier towards the choroid, play a crucial role in supporting and maintaining the photoreceptors. Their tasks include active transport of nutrients from the choroidal vessels to the photoreceptors, processing of vitamin A, and uptake and recycling of outer segments which are continuously shed by the photoreceptors. While primary degeneration of the photoreceptors is the cause of progressive visual loss in some types of Retinitis Pigmentosa, in others the initial injury is in RPE cells, and as a consequence, the photoreceptors are damaged as well and retinal degeneration ensues.
  • Age-Related Macular Degeneration (AMD) 5 which is the most common cause of blindness among the elderly aging population, failure of the RPE is the main cause of disease [Smith W, Assink J, Klein R et al. Risk factors for age-related macular degeneration: Pooled findings from three continents. Ophthalmology 2001; 108:697-704.
  • hESC-derived RPE cells may serve for high throughput screening for compounds that are toxic to RPE cells. They may be used to uncover mechanisms, new genes, soluble or membrane-bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells. These cells may serve as an unlimited source of
  • RPE cells in retinal degenerations may serve as a vector to carry and express genes in the retina after transplantation.
  • hESC-derived RPE cells were transplanted into the vitreous and into the subretinal space of RCS and albino rats respectively. Immuno-histological analysis of the eyes, at 4 weeks after transplantation, demonstrated . surviving hESC-derived RPE cells ( Figure 32A-32B). Transplanted RPE cells migrated from subretinal grafts and integrated within the RPE layer of host albino rats ( Figure 32C).
  • a differential comparison of the gene expression profile of EBs after 4 weeks of suspension culture in the presence and absence of NA was performed by using affymetrix gene arrays.
  • the expression level of 1072 genes was up- or down- regulated by at least two-fold; 442 genes were up-regulated, and 630 genes were down- regulated.
  • the analysis confirmed that NA suppressed the expression of ⁇ -fetoprotein and cytokeratin-8.
  • the expression of other cytokeratins was also suppressed, including cytokeratins 7, 18, 19, 23.
  • chordin-like-1 gene was up-regulated. This finding was further confirmed by RT PCR ( Figure 31). Chordin-like is composed of three cysteine-rich (CRs) domains. Chordin-like binds BMP -4,
  • chordin expression is restricted to the node and the notochord at a time in which chordin-like is expressed in the neural plate [Garcia Abreu, J., et al.
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WO2006070370A3 (en) 2007-03-08
EP2410044A3 (en) 2013-03-13
EP2410044B1 (en) 2022-06-22
US20090104695A1 (en) 2009-04-23
EP2410044A2 (en) 2012-01-25
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US20110177594A1 (en) 2011-07-21
EP2410043A3 (en) 2013-01-23

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