Home > Boards > US OTC > Biotechs > Protide Pharmaceuticals (PPMD)

This is a cut and paste from the

Public Reply | Private Reply | Keep | Last ReadPost New MsgReplies (1) | Next 10 | Previous | Next
Lazarus Member Profile
Member Level 
Followed By 117
Posts 5,402
Boards Moderated 1
Alias Born 04/26/01
160x600 placeholder
Lazarus Member Level  Monday, 10/29/07 03:18:03 PM
Re: None
Post # of 569 
This is a cut and paste from the RB board...

(courtesy chuuk333)

World Intellectual Property Organization

(Look down to the last couple of paragraphs)

Notice who produces the other product that didn't impair undifferentiated stem cell propagation? ROCHE!!!
So there are big names in this business-and hopefully are little charmers at PPMD will catch someones eye some day.

--------------------------------------------------------------------------------

(WO/2006/070370) STEM CELLS CULTURE SYSTEMSBiblio. Data
Description
Claims
National Phase
Notices
Documents
Note: OCR Text
Note: Text based on automatic Optical
Character Recognition processes. Please
use the PDF version for legal matters


STEM CELLS CULTURE SYSTEMS

FIELD OF THE INVENTION

The invention relates to stem cells (SC) in particularly to methods and systems for handling human embryonic stem cells (hESC).

LIST OF PRIOR ART

The following is a list of prior art, which is considered to be pertinent for describing the state of the art in the field of the invention.

(1) Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147 (1998).

(2) Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399-404 (2000)

(3) Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.

Dev Biol 227, 271-278 (2000).

(4) Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19, 971-974 (2001).

(5) Amit, M. et al. Human feeder layers for human embryonic stem cells. Biol Reprod βS, 2150-2156 (2003).

(6) Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C. & Bongso, A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20, 933-936 (2002).

(7) Cowan, CA. et al. Derivation of embryonic stem-cell lines from human blastocysts. N EnglJ Med 350, 1353-1356 (2004).

(8) Amit, M., Shariki, C, Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and Serum-Free Culture of Human Embryonic Stem Cells. Biol Reprod 70(3):837-45 (2004).

(9) Pera, M.F. et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci 111, 1269-1280 (2004).

(10) GB2409208.

(11) WO 04/031343

(12) Xu, R.H., et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 3, 164-5 (2005)

(13) Valuer L, et al. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci. 118, 4495-509 (2005)

BACKGROUND OF THE INVENTION Stem cells are immature, unspecialized cells that renew themselves for long periods through cell division. Under certain conditions, they can differentiate into mature, functional cells. Human embryonic stem cells (hESC) are derived from early surplus human blastocysts l' 2. Human 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 body1' 2.

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. Since 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. In addition, transplanted genetically- modified hESC can serve as vectors to carry and express genes in target organs in the course of gene therapy.

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.

At present, few culture systems are most commonly used to propagate undifferentiated hESC1"4. In the initial culture system that was developed, undifferentiated hESC are cultured in serum-containing medium as colonies, upon a layer of fibroblast feeder cells (of mouse1' 2 or human origin5' π). It is possible to remove all animal products from this culture system and replace them with those from a human source6. It was found that in this system the cells are propagated as clumps on a low scale which does not allow cloning2.

An alternative culture system that was developed and used extensively is a serum-free system that includes the knockout (KO) medium supplemented with knockout serum replacement (KOSR) and FGF2. This system allows cloning of undifferentiated hESC, although at a low efficiency3. Undifferentiated cells are cultured as flat colonies and may be propagated as small clusters or single cells (by using trypsin7).''

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. 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).

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 FGF24. Furthermore, it has been suggested that feeder conditioning may be replaced by substituting the medium with high concentrations of FGF2 and noggin12. Alternatively, feeder conditioning was replaced by transforming growth factor β 1 and human LIF (in addition to FGF2) and growing the cells on human fibronectin . In a recent publication, 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 .

A key limitation of hESC culture systems is that they 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 endoderm9.

Spontaneous' differentiation of hESC into presumably extraembryonic lineages also interferes with the derivation of somatic differentiated cells. Under various differentiation-inducing conditions, such as in embryoid bodies (EB) suspension cultures, differentiation into cystic extraembryonic structures may be common or may predominate and limit differentiation into somatic lineages.

Control and elimination of the differentiation into extraembryonic lineages therefore, may be invaluable in the derivation of somatic lineages, in addition to its importance in maintaining the stem cells in an undifferentiated state. It has been recently demonstrated that under differentiation-inducing culture conditions, the bone morphogenetic protein (BMP) antagonist noggin can prevent extraembryonic differentiation of hESC and promote their differentiation into the neural lineage9.

SUMMARY OF THE INVENTION

In accordance with a first aspect, 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. According to one preferred embodiment, the culture system comprises the human umbilical cord derived feeder cells of the invention.

Within this aspect of the invention there is also provided 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.

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.

In accordance with a second aspect, 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. 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.

In accordance with this second aspect, there is also provided a method for inhibiting or preventing differentiation of stem cells to extraembryonic cells, the method 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.

Further in accordance with this aspect of the invention there is provided the use of 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.

In yet a third aspect of the invention there is provided 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.

In accordance with this aspect there is also provided 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.

In accordance with this aspect of the invention, there is also provided a method of maintaining stem cells in an undifferentiated state, the method comprises incubating said cells with a culture system comprising animal free stem cell basic culture medium and humanized serum replacement substitute.

In accordance with a fourth aspect of the invention there is provided a culture system for maintenance SC in an undifferentiated state, the culture system comprising Neurobasal™ medium.

Within this aspect there is also provided a culture of SC in an undifferentiated state, the SC culture being obtained by culturing a cluster of cells from inside a blastocyst with a culture system comprising Neurobasal™ medium.

In accordance with this aspect of the invention there is also provided a method for maintaining a culture of SC in an undifferentiated state, the method comprising incubating said cells with a culture system comprising Neurobasal™ 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 Neurobasal™-based culture system in the form of a suspension as well as in a monolayer (flat colonies). Preferably, the Neurobasal™ medium is supplemented with N2 supplement or an N2 like supplement as defined below.

Finally, there is provided in accordance with the invention a method of maintaining SC in an undifferentiated state comprising culturing SC with feeder cells expanded from human umbilical cord cells.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non- limiting example only, with reference to the accompanying drawings, in which:

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

SSEA4, TRAl-60, TRA1-81 and SSEAl by hESC on cord derived feeders

(Figs. 6A-6D, respectively), by hESC on human embryonic fibroblasts (Fig. 6E-

6H5 respectively) or by hESC on foreskin (Fig. 6I-6L, respectively).

Figure 7A-7C - are immunofluorescent images of hES colonies expressing AP5 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).

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 (2W5 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

CORD2), 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 2, 3, 7 and

10 weeks (2W, 3W5 7W and 10W).

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

(IW, 2W5 5W and 9W).

Figure 14 - is a bar graph representing analysis of the percentage of hESC cultured on two independent human embryonic fibroblast cell lines (HEFGl and

HEFG2), 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 and 6 weeks (IW and 6W).

Figures 15A-15I are immunofluorescent images of EBs-derived differentiated cells expressing β-tubulin (Figs. 15A5 15D and 15G)5 AFP (Figs. 15B5 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 SR5 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. 25A-25D, i.e. upper panel). In the presence of NA, cystic formation was not observed, the EBs were comprised of tightly packed cells and were significantly larger (Figs. 25E-25H, i.e. second panel). In the presence of RA, the EBs were smaller and included multiple cysts (Figs. 25I-25L, i.e. third panel). NA blocked the effects of RA (Figs. 25M-25P, i.e. lower panel).

Figure 26 - is a RT-PCR analysis demonstrating that the expression of the endodermal marker α-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 α-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-α-fetoprotein (Fig. 27A) and cytokeratin-8 (Fig. 27B). Cells that expressed α-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. Figures 30A-30H -_are images showing that melanin-expressing cells that were generated in the presence of NA had morphological characteristics that are typical of RPE cells. Specifically shown are dark field micrograph (Fig. 30A), phase contrast image (Fig. 30B) and indirect immunofluorescent stainings of RPE cells markers including ZO-I (Fig. 30C), Pax6 (Fig. 30D), MITF (Fig. 30E), CRALBP (Fig. 30F), Bestrophin (Fig. 30G) and RPE65 (Fig. 30H).

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.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

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.

As used in the specification and claims, the forms "a", "an" and "the" include singular as well as plural references unless the context clearly dictates otherwise. For example, the term "a culture system" includes one or more culture systems.

As used herein, the term "or" means one or a combination of two or more of the listed choices Further, as used herein, the term "comprising" is intended to mean that the methods or composition includes the recited elements, but not excluding others. Similarly, "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. For example, 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. Also, a composition 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.

Further, all numerical values, e.g., concentration or dose or ranges thereof, are approximations which are varied (+) or (-) by up to 20%, at times by up to 10% of from the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

In its broadest sense, the present invention concerns culture cells, systems and methods for use of same in culturing of stem cells. As used herein, the term "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.

As used herein, 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. Furthermore, as used herein, 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 phenόtype), primary cell cultures, finite cell lines (e.g., non- transformed cells), and any other cell population maintained in vitro.

As used herein, the teπn "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. Preferred stem cells according to the present invention are human stem cells, more preferably, hESC.

Stem cells can be obtained using well-known cell-culture methods. For example, 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. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells 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. Following 9 to 15 days, 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. 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. USA 92: 7844, 1995]; as well as Bongso et al, [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69:84, 1998].

Commercially available 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.

Potential applications of hESC are far ranging and include drug discovery and testing, generation of cells, tissues and organs for use in transplantation, production of biomolecules, testing the toxicity and/or teratogenicity of compounds and facilitating the study of developmental and other biological processes. For example, 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]. There are, however, significant hurdles to the practical exploitation of hESC. To maintain hESC in an undifferentiated pluripotential state, the cells are usually cultured on feeder cells. The 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).

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].

While widely used, human SC cultures based on murine derived feeder cells, are less desired. 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. For example, such non-species specific feeder cells contain both foreign cells and foreign growth factors. Further, it is believed that 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. It has been shown that 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. Thus, 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 (either the feeder cells or the SC cultures) in accordance with the invention may be a fresh cell culture, cryopresrved culture as well as cryopreserved and thawed cells.

As used herein, the term "derived" which may be used interchangeably with the term "obtained" when used in the context of cell formation denotes the development of a new cell line from another cell line. For example, 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.

Further, as used herein, the term "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. Specifically, 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). Generally, the feeder cells may be fibroblasts or other types of cells, and the cells are inactivated by large- dose radiation before use, such as γ-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.

As indicated above, the feeder cells are derived (expanded) from umbilical cord tissue. Umbilical cord tissue may be obtained in the course of vaginal delivery. However, 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. Moreover, 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.

An additional advantage of 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.

In this connection there is thus also provided a method for preparing umbilical cord derived feeder cells, the method 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.

In accordance with the invention there is also provided a culture system for maintaining stem cells (SC) in an undifferentiated state, the culture system 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".

As used herein with respect to all aspects 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. "Proliferation", "propagation" ,

" expansion" and "growth", which are used interchangeably, refer to such an increase in cell number. According to one embodiment, when referring to maintenance of hESC on feeder 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.

When the feeder cells are derived from human umbilical cord tissue, 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.

In accordance with one embodiment, 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. As used herein, 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. Preferably, the conditions are such which enable growth of human stem cells, preferably, hESC. Further, 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. It is not intended that the term "culture medium" or "nutrient medium" be limited to any particular culture medium. For example, it is intended that the definition encompass outgrowth as well as maintenance media. In accordance with the human derived feeder cell aspect of the invention, the culture system also comprises the feeder cells. However, 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.

In accordance with one embodiment, 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. 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

Medium (DMEM), 20% defined Fetal Calf Serum, 1% L-Glutamine, 0.5% penicillin/streptomycin, 1% non-essential amino acids, 1% Insulin-Transferrin- Selenium G supplement and 1 mM β-mercaptoethanol.

In an animal free culture system, which provides a pathogen-free environment for the growth of ES cells, 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. Paul, MN, or equivalent) or Nutridoma-CS (Roche, Germany, or equivalent). Since the animal free system provides a pathogen free environment, reducing agents such as β-mercaptoethanol and antibacterial agents such as penicillin/streptomycin) may be eliminated. In the context of the human derived feeder cell culture system aspect of the invention there is also provided a method for maintaining stem cells in an undifferentiated state, 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.

According to one embodiment, 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 370C and 5% CO2 in air are generally adopted.

In cultures of undifferentiated hESCs there is always some level of background extraembryonic differentiation. Further, in currently-used systems for the cultivation of undifferentiated hESCs, or for induction of their differentiation towards somatic lineages, three is tendency of hESCs to differentiate towards extraembryonic lineages. In addition, upon induction of differentiation, the default pathway of differentiation towards extraembryonic lineages may predominate, and limit differentiation into desired somatic lineages.

Thus, 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. 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).

The term "derivative of nicotinamide" as used herein denotes a compound which is a chemical modification of the natural NA.


Nicotinamide (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). A variety of 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]. For example, the process of preparing 4-aryl-nicotinamide derivatives are described in WO05014549A. 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.

The effect of 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".

In accordance with the NA aspect of the invention, 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. As used herein "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.

Proliferation and differentiation of embryonic stem cells into insulin- producing cells in the presence of NA was suggested by Vaca P .et al,

[Transplant Proc. 35(5):2021-3 2003] Specifically, it was shown that while proliferation within EBs with or without supplementation of the medium with

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.

It has now been found that NA effectively induced differentiation of stem cells into somatic cells. Specifically, albeit, not exclusively, NA was shown to induce differentiation to neural cells, and within the neural lineage, NA treatment was found to promote differentiation towards retinal pigmented epithelial (RPE) cells. The use of RPE cells in transplantation has already been described [Haruta, M. et al, In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells. Invest Ophthalmol Vis Sci, 45:1020-1025 (2004)]. Thus, it is to be understood that the RPE cells obtained in accordance with the present invention have various therapeutic applications. One such application includes transplantation of such cells in the eye to replenish malfunctioning or degenerated RPE cells in retinal degenerations. Genetically modified 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. The 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.

In the context of this aspect of the invention there is also provided a method for inhibiting or preventing differentiation of stem cells towards extraembryonic lineages (to extraembryonic cells), the method comprises incubating said stem cells in a culture system comprising NA or a derivative of

NA as defined above.

It should be noted that in the context of the present invention the 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.

It should also be noted that the NA based culture system of the invention was effective to induces an increase in number of cells within embryoid bodies (EB) cultured therein. For induction of somatic differentiation, the stem cells in accordance with the NA aspect of the invention are preferably grown as free floating clusters in a suspension. As used herein, 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. Lot-to-lot variation is often observed and some lots of serum have been found to be toxic to cells [Robertson, EJ., ed., Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, IRL Press, Oxford, UK (1987)]. Moreover, serum may be contaminated with infectious agents such as mycoplasma, bacteriophage, and viruses. Finally, because serum is an undefined and variable component of any medium, the use of serum prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells.
?????????????????????????????????????????????????????????????????????
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
It has now been found that the use of two well known and commercially available humanized serum replacement (SR) substitutes, which have not been used hitherto in human embryonic stem cell technology, i.e. TCH™ (Protide Pharmaceuticals, St. Paul, MN, or equivalent) and Nutridoma-CS (Roche, Germany, or equivalent) used as serum replacement substitute, did not impair the undifferentiated propagation of the stem cells in the culture system.

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
??????????????????????????????????????????????????????????????????????
Thus, according to a further aspect, 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. This aspect of the invention is referred to as the "humanized serum free culture system of the invention".

In accordance with one embodiment, 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,

Neurobasal™, or X-Vivo 10.

In accordance with another embodiment, the humanized culture system comprises a serum replacement substituent selected from TCH™, Nutridoma-CS or combination of same.

In accordance with yet another embodiment, when said serum free basic medium is Neurobasal™, 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. It is noted that the standard and commercially available N2 supplement comprises insulin, transferrin, progesterone, putrascine, selenite. The specific composition of 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), 0.52μg/mL sodium selenite, 1.61mg/mL putrascine, 0.63μg/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. For example, medium for the propagation of ESC-derived neural stem cells is supplemented with modified N2 [Conti, L, et al, Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol. 9:e283 (2005)]. TCH™ is a completely biochemically defined serum replacement developed primarily for human cells and production of cell-secreted proteins. TCH™ may be purchased from Protide Pharmaceuticals (MN, USA) as well as from BM Biomedicals (CA, USA).



Public Reply | Private Reply | Keep | Last ReadPost New MsgReplies (1) | Next 10 | Previous | Next
Follow Board Follow Board Keyboard Shortcuts Report TOS Violation
X
Current Price
Change
Volume
Detailed Quote - Discussion Board
Intraday Chart
+/- to Watchlist
Consent Preferences