FORM 2
THE PATENTS ACT, 197 (39 of 1970)
AND
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION (See Section 10, rule 13)
ESTABLISIINT OF A HUMAN EMBRYONIC SUM CELL LNE USING
MAMMALIAN CELLS

RELIANCE LIFE SCIENCES PVT.LTD.
an
Indian Company tag its Registered office at
Chitrakoot, 2nd Floor, Ganpatrao Kadam Marg,
Shree Ram Mills Compound, Lower Parel, Mumbai 400 013, Maharashtra, India

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

59

MUM

FIELD OF THE INVENTION

The present invention relates to the isolation, maintenance and propagation of human embryonic
stem cell line (hE8C) fom the inner cell mass (ICM) of the surplus.. This disclouser also reltes
to the characterization of the hES cell line thereby demonstrating its in vitro differentiation potential and its prospective use in cell therapy and drug screening.
BACKGROUND OF THE INVENTION
Human pluripotent Stem Cells have been derived from inner cell mass (ICM) and primordial germ cells of developing gonadal ridge (embryonic germ cells) (Gearhart, 1998).
Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of the mammalian blastocyst (Evans & Kaufman 1981; Martin 1981). These cells are pluripotent, thus capable of developing into any organ or tissue type. ES cells are capable of indefinite proliferation in vitro in an undifferentiated state; maintaining a normal karyotype through prolonged culture; and maintaining the potential to differentiate into derivatives of all three embryonic germ layers (i.e., mesoderm, ectoderm and endoderm) (J. Itskovitz -Eldor et al. 2000).
Embryonic stem cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo as well as providing opportunities for genetic manipulation. Appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells, suitable for cell based therapies of diseases that result from cell damage or dysfunction.
Embryonic stem cells have been isolated from the inner cell mass (ICM) of blastocyst stage embryos in mice (Solter and Knowles, 1975) and other species. Early work on ES cells was done in mice (Doetschman et al., 1985). Mouse ES (mES) cells are undifferentiated pluripotent cells derived in vitro from preimplantation embryos. mES cells maintain an undifferentiated state

Although mouse ES cells facilitate the understanding of developmental processes and genetic
diseases, significant differences in human and mouse developent limit the use of mouse ES
cells as a mode of human development. The morphology, cell surface markers and growth requirement of embryonic cells from other species are significantly different from mouse ES cells. Further, mouse and human embryos differ significantly in temporal expression of embryonic genes, such as in the formation of the egg cylinder versus the embryonic disc (Kaufman, The Atlas of Mouse Development;London;Academic Press, 1992) in the proposed
derivatioii of some early lineages (O'Rahilly and Muller; Develomental stages In Human
Embroys Washington Carenige Institution of Washington, 1987) in the structure and function
of the extraembryonic membranes and placenta (Mossman, Vertebr ate Fetal membranes; New Brunswick; Rutgers, 1987) in growth factor requirement for development (e.g., the hematopoietic system- Lapidot Lab. Animal Sciences 1994) and in adult structure and function (e.g., central nervous system). To overcome this and to have a better insight into human embryonic development, ES cells have been successfully established from primates (Thomson et
a!.,1995 and 1998)
The cell lines currently available that resembles human ES cells most closely are human embryonic carcinoma (EC) cells, which are pluripotent, immortal cells derived from teratocarcinomas (Andrews, et al., Lab. Invest. 50(2):147-162, 1984; Andrews, et al., in: Robertson E., ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL press, pp. 207-246, 1987). EC cells can be induced to differentiate in culture, and the differentiation is characterized by the loss of specific cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and the appearance of new markers (Andrews, et al., 1987, supra). Human EC cells will form teratocarcinomas with derivatives of multiple embryonic lineages in tumors in nude mice. Similar mouse EC cell lines have been derived from teratocarcinomas, and, in general their developmental potential is much more limited than mouse ES cells (Rossant, et al., Cell Differ. 15:155-161, 1984). Teratocarcinomas are tumors derived from germ cells, and although germ cells (like ES cells) are theoretically totipotent (i.e. capable of forming all cell types in the body), the more limited developmental potential and the abnormal karyotypes of EC cells are thought to result from selective pressures in the teratocarcinoma tumor environment (Rossant & Papaioannou, Cell Differ 15:155-161, 1984). ES cells, on the other hand, are thought

to retain greater developmental potential because they are derived from normal embryonic cells in vitro, without the selective pressures of the teratocarcinoma environment. Pluripotent cell lines have also been derived from preimplantation embryos of several domestic and laboratory animals species (Evans, et al., Theriogenology 33(1):125-128, 1990; Evans, et al., Theriogenology 33(1):125-128, 1990; Notarianni, et al., J. Reprod. Fertil. 41(Suppl.):51-56, 1990; GileS, et al, MOl, faprod, Dev. 36:130-138, 1993; Graves, et al., Mol. Reprod. Dev 36:424-433, 1993; Sukoyan, et al., Mol. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al., Mol. Reprod. Dev. 36:148-158,1993; Iannaccone, et al., Dev. Biol. 163:288-292, 1994).
The first human pluripotent embryonic stem cell line was established and named HI, H7, H9, HI 3, and H14 by Dr. Jamie Thomson and his co-workers, at the University of Wisconsin in the United States (Thomson, J. A. et al 1998, Science 282:1145-1147.) A few years later, Dr. Martin J. Pera and coworkers reported having established human embryonic stem cell lines ("ES cell lines"), which were named HES-1 and HES-2, from human blastocysts.( Reubinoff, B. E. et al. 2000, Nat. Biotechnol. 18:399-404). To date, the majority of described hES cell lines were derived from day 5 to day 8 blastocysts produced for clinical purposes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Although, the isolation of ICM from morula (day 4 embryo) stage has also been reported (Giles et al., 1993). One of the hES cell lines has been derived from blastocysts developed from embryos reconstructed using a somatic cell nuclear transfer (SCNT) technique (Hwang et al., 2004).
Human embryonic stem cells (hES) can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo pre-implantation embryos or from in vitro fertilized (IVF) embryos, intracytoplasmic sperm injection, ooplasm transfer, or other ART methods well known to those of skill in the art. Human ES cells may be derived from a blastocyst using standard immunosurgery techniques as disclosed in U.S. Patent Nos. 5,843,780 and 6,200,806, Thomson et al. (Science 282:1145-1147, 1998) and Reubinoff et al. (Nature Biotech. 18:399-403, 2000), or by a unique method of laser ablation (U.S. Serial No. 10/226,711 each specifically incorporated herein by reference. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. Although numerous human ES cell lines have been derived till date, only a few of them are well characterized (Brimble et al., 2004) in terms of their unique identity, self renewal capacity and differentiation potential. The inventors of the present invention have been
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successful in establishing a well characterized cell line in terms of their unique identity, self renewal capacity and differentiation potential.
Human ES cells are isolated by removing zona-pellucida from the blastocyst and the inner cell MSS (KM) IS isolated by immunosurgeryl) in Which the trophectoderm cells are lysed and
removed from the intact ICM by gentle pipetting, The ICM \s 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 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 week (Thomson et al., [U.S. Pat. Mo. 5,843,780; ScifittCe lfo 1145, 1998; Cliff. TOP. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706,1989]; Gardner et al., [Fertil. Steril. 69: 84,1998).
Human ES cells are maintained in an undifferentiated pluripotent state in the presence of a feeder layer or feeder free condition on a extra cellular matrix supplemented with serum or conditioned medium. The feeder layers are either y irradiated or mitomycin-C treated mouse embryonic fibroblast (MEF) cells. When cultured in a standard culture environment in the absence of MEF as feeder cells the human embryonic stem cells rapidly differentiate or fail to survive. Unlike murine embryonic stem cells, the presence of exogenously added LIF does not prevent differentiation of human embryonic stem cells. Feeder cell layers are used to provide a microenvironment (or niche) to prevent stem cells from differentiating along their natural course. Feeder tissues provide the stem cells with external signals such as secretion of factors and cell-to-cell interactions mediated by integral membrane proteins. (Watt F. M. and Hogan L. M. (2000) Science 287:1427-1430.) In light of the fact that secretion factors and direct cell-to-cell interactions control in vitro survival, proliferation, and differentiation of the stem cells, an ideal environment should consist of healthy feeder tissues with normal microstructures and functions.
Examples of feeder cells are: (1) irradiation-inactivated mouse embryonic fibroblasts; (2) mitotically (mitomycin C) inactivated mouse embryonic fibroblasts; and (3) irradiation-

inactivated STO fibroblast feeder layers. (Thomson, J. A. et al, (1998); Reubinoff B. E. et. al. (2000); and Shamblott, M. J. et al. (1998); Proc. Natl. Acad. Sci. U.S.A. 95:13726-13731.
In spite of this progress, several significant disadvantages still exist. Exposure to animal pathogens through MEF conditioned medium or matrigel matrix is still a possibility. The major obstacle of the use of hES cells in human therapy is that the originally described methods to propagate them involved culturing on a layer of feeder cells of animal origin. In recent years, extensive research into improving culture systems for hES cells has been concentrated in the ability to grow cells under serum free/feeder free conditions. To ensure a feeder layer free environment for the growth of hES cells, a substitute system based on medium supplemented with serum replacement (SR), transforming growth factor Bl (TGF-B1), LIF, bFGF and a fibronectin matrix has also been tried (Amit et al 2004: Biol. Reprod. 70, 837-845). However, continuous evaluation of methods for derivation and propagation of undifferentiated hES cells on human feeders or feeder free matrices to obtain pure cultures of hES cells without contamination of cells and proteins from other species has not been successful.
The detailed characterization of hES cells include their analysis at cellular and molecular levels, regulation of cell cycle, expression of high telomerase activity, genetic stability, particular HLA and STR type and differentiation potential under in vitro and in vivo conditions. The profile of surface antigens displayed in undifferentiated hES cells matches that of human ES cells and human embryonal carcinoma (EC) cells. Undifferentiated hES express globo-series cell surface markers: stage specific embryonic antigen SSEA-3, SSEA-4, as well as tumor recognition antigens TRA-1-60 and TRA-1-81. The expression of POU5F1 promoter encoded transcription factor OCT-4, E-cadherin and gap junction protein connexin 43 are detected (Andrews et al., 2002). Unlike mouse embryonic stem cells, undifferentiated human embryonic stem cells do not express SSEA-1.Undifferentiated hES cells stain positively for alkaline phosphatase and demonstrate high telomerase activity indicative of their increased self-renewal capacity.
The genetic stability of the hES cells can be assessed by using standard G-banding technique. Normally hES cells maintain a stable karyotype either 46 XX or 46 XY even after prolonged continuous culture. However with increased passaging the cells tend to show abnormal karyotype, trisomies of chromosomes 12-17 and X chromosome may occur.
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unlimited proliferate potential of ES cells is directly correlated with telomerase activity.
Telomerase Repeat Amplification Protocol (TRAP) assay is usually performed to assess the telomerase activity. The assay can be performed either using a radioisotopic method (Thomson et al., 1998) or a non-radioisotopic method (Oh et al., 2004).
Human cells have the potentail differentiate into all the 210 cell types of the human body.
The developmental potential of these cells after prolonged culture are examined in vitro by the
formation of embryoid bodies and in vivo by the formation of teratomas in SCID mice (Evans M J and Kaufman M, 1983). To confirm that hES cells retain their in vitro differentiation capacity, embryoid bodies can be formed in suspension culture and analysed by RT-PCR and immunocytochemistry for markers representing each of the three germ layers (J. Itskovitz-Eldor, 2000 and Shamblott et al., 1998).
Human ES cells offer insight into developmental events, which cannot be studied in explant systems. Screens based on the in vitro differentiation of human ES cells to specific lineages can identify gene targets, which can be used for designing and reprogramming of tissue regeneration and teratogenic or toxic compounds. Replacement of non-functional cells using ES cells technology can offer a permanent treatment in case of degenerative diseases like Parkinson's disease, stroke, cardiac ischemia, hepatic failure, juvenile-onset diabetes mellitus which result from the death or dysfunction of one or several cell types (Wobus and Boheler, 2005). However, therapeutic application of human ES cells requires defined growth conditions, pathogen-free environment and survival at extended in vitro conditions, to enable enriched production of ES derived specialized cell types.
Recently, the application of stem cells in toxicology has been reported (Davila et al., 2004). The overwhelming benefit of stem cells, when applied to toxicology, evolves from their unique properties compared to primary human cells, i.e. unlimited proliferation ability, plasticity to generate other cell types, and a more readily available source of human cells. Currently, in vitro differentiation of mouse ES cells to hepatocytes has also been reported (Hamazaki et al., 2001; Tones et al.. 2002). However the utility of these differentiated hepatocytes, as an in vitro

Results on mouse ES cells demonstrate that hepatocytes generated from mouse ES cells
may prove to be a suitable adjunct to the conventional in Vitro toxicity models for drug
metabolism and toxicity studies.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspect disclosure, the inventions of which can be better
understood by reference to one or more of these drawings in combination with the MH
description of specific embodiments presented herein.
Figure 1 represents photomicrographs of the human blastocyst at low (100X) and high (200X) magnification, which was used for the establishment of Relicell™hESl cell line. Photograph 1.2 show a day 6 blastocyst with a clearly visible zona-pellucida, mono-layered trophectoderm and
a poorly developed inner cell mass (ICM). The embryo was assigned as Grade-C.
Figure 2 are photographs showing the qualification of mouse embryonic fibroblast (MEF) cells used for the culture and propagation of Relicell™hESl cell line. Photograph 2.1 represents a photomicrograph of MEF cells showing 80% confluency, 48 hours post plating; Photograph 2.2 demonstrates a healthy hES cell colony grown on MEF cells; Photographs 2.3 and 2.4 shows positive immunostaining of hES cells on MEF with Oct-3/4 and SSEA-4 antibodies; Photograph 2.5 demonstrates the expression of ES cell markers in hES cells grown on MEF cells.
Figure 3 represents a set of phase-contrast photomicrographs demonstrating the morphology of Relicell™hESl cells at progressive days of plating upon MEF layers in a media containing human LIF (10ng/ml). The ICM cell mass is seen to have attached after day 1 of plating, which gradually expand on the MEF cells upto 12 days. At day 12, a hES cell colony is formed and then passaging of the colonies are performed to propagate the cell line.
Figure 4 are a set of phase-contrast photomicrographs demonstrating the morphology of the undifferentiated hES cell colonies at different passages starting from passage 10 upto passage 30. Photograph 3.1 shows a compact hES cell colony on healthy looking MEF cells, which not only provides nutrition to these ES cells but also facilitates in maintaining them in the undifferentiated
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state. Photograph 3.4, taken at a higher magnification (200X) demonstrates the distinct cell borders, high nucleus to cytoplasm ratio, and prominent nucleoli or undifferentiated human ES
Cells, ES cells M maintained in a medium comprising of DMEM supplemented with 10% FBS
and human LIF (10ng/ml).
Figure 5 represents a set of eight photomicrographs showing phenotypic expression of different ES cell markers detected by immunocytochemistry for cells grown with primary feeder cells (MEF). Photograph 5.1 shows Oct-3/4 (+); Photograph 5.2 shows SSEA-3 (+); Photograph 5.3
SHOWS SSEA (+Photograph 5,4 shows Tra-1-6O (+) Photograph 5.5 shows Tra-1-81 (+);
Photograph 5.6 shows Connexin-43 (t); Photograph 17 shows E-cadherin (t) and Photograph 18 shows Alkaline phosphatase (t) immunostaining with hES cells fixed in 4%
paraformaldehyde. All the markers analyzed are carbohydrate rich cell surface antigens except Oct-3/4, which is a PoU5Fl promoter encoded transcription factor and Connexin-43, a gap junction molecule. The immunofluorescence analysis is carried out at every 5th passage of the propagation Of R9li99ll™hESl and all antibodies used were FITC labeled.
Figure 6 is a photograph illustrating the expression of undifferentiated genes including Oct-4, Nanog, Rexl, Sox2, Thyl, FGF4, ABCG2, Dppa5, UTF1, Criptol, hTERT, Connexin-43 and Connexin-45 in the hES cell line at passage 22, thereby establishing the pluripotency of the hES cells. The enhanced expression of hTERT gene is indicative of the high self-renewal capacity of the hES cell line. GAPDH, is used as a housekeeping gene control. Lane 1 and Lane 17 show the standard, which is a 100bp ladder, the lowest band being of 100bp. The RT-PCR analysis is carried out at every 5th passage of the propagation of Relicell™hESl. For details of the primers please refer Table 1.
Figure 7 represents a 15% acrylamide gel picture, showing substantial telomerase activity of ReliCellnc]hESl at passage 37, using PCR based SYBR green staining. 6ug of total protein was loaded for each assay. Lane 1: NTERA-2; Lane 2: NTERA-2 (Heat inactivated); Lane 3: MEF; Lane 4: MEF (Heat inactivated); Lane 5: ReliCellDDhESl (p37); Lane 6: ReliCellDDhESl (p37, Heat inactivated); Lane 7: Primer-Dimer control; Lane 8: TSR8 control template (1.5ul, provided

Figure 8 includes four photographs of embryoid bodies at increasing days in $U$
maintained in a suitable medium (w/o hLIF), to induce differentiation in vitro. Photograph s.l shows loose aggregate/colony of hES cells after 6 days in suspension culture; Photograph 8.2 shows a compact embryoid body at day 10; Photograph 8.3 demonstrates the initiation of blood island formation at day 14 and Photograph 8.4 shows dense formation of blood islands at day 21,
which is the evidence of angiogenesis in VitrO,
Figure 9 are photomicrographs demonstrating the in vitro differentiation potential of Relicell™hESl by immunochemistry of the fixed embryoid bodies (day 14) in 2 well-chamber slides. Photograph 10.1 shows nestin (+) immunostaining (ectoderm); Photographs 10.2 & 10.3 shows smooth muscle actin & brachyury (+) immunostaining (mesoderm) and Photographs 10.4 ft 10 5 5llOW§ AFp & GATA-4 (+) immunostaining, thereby confirming the RT-PCR results. All
antibodies used for the study were FITC conjugated. Pictures were acquired in Nikon E600 inverted microscope.
Figure 10 shows the differential gene expression of a set of lineage specific markers responsible for development of three germ layers including Keratin 8, Keratin 15, Keratin 18, NFH, Sox-1 (ectoderm), Brachyury, MyoD, Msxl, HAND1, cardiac actin (mesoderm) and GATA-4, AFP, HNF-3b, HNF-4a, albumin, PDX1 (endoderm) in embryoid bodies (passage 32) generated from Relicell hESl. The photograph demonstrates high mRNA levels of the aforesaid markers at day 10 upto day 14 of embryoid body formation, thereby indicating in vitro differentiation potential of the hES cell line into all three lineages. HEF cells are used as a negative control and GAPDH is used as a housekeeping gene control. For details of the primers please refer Table 1.
DETAILED DESCRPITION OF THE INVENTION
The present disclosure relates to the isolation of human ES cells from the inner cell mass of blastocyst stage of mammalian embryo by immunosurgery. In preferred embodiments, the pluripotent ES cells are capable of self-regeneration and can give rise to cells of all the three lineages including as ectoderm, mesoderm and endoderm.

In other preferred embodiments of the present disclosure the isolated blastocyst from which cells
of the inner cell mass (ICM) are isolated using immunosurgery are produced by In vitro
fertilization, intracytoplasmic sperm injection, ooplasm transfer, or other ART methods well known to those of skill in the art.
In the present invention, several methods including but not limited to immunosurgery, micro surgery laser ablation technique can be used to isolate cells from the ICM for the
establishment of human embryonic stem cell lines.
In the preferred embodiments, the hES cells were grown on embryonic fibroblast cells including but not limited to mouse embryonic fibroblasts, human embryonic fibroblasts or fibroblast-like cells derived from adult human tissues.
In preferred embodiments, the cells of the present disclosure were cultured and maintained by manual passaging in a media containing 80% DMEM/F-12, 15% ES tested FBS, 5% Serum replacement, 1% nonessential amino acid solution, ImM glutamine (Gibco), 0.1% beta mercaptoethanol, 4ng/ml human bFGF and 10ng/ml human Leukemia inhibitory factor. The method of manual passaging as disclosed in the present invention is advantageous over the commonly used method of passaging by enzymatic treatment, for retaining the genetic stability of the cell line. Maintenance of the normal karyotype of a cell line is important for its usage in therapeutic purposes.
Generally MEFs used for maintenance and passaging of hESC lines are obtained from commercial sources. However we have been successful in deriving MEF's inhouse by a process as stated herein. The mouse feeder layers used in the present disclosure, were derived in-house by the following method: 1) Procurement of pregnant mice and dissection; 2) Staging of mice embryos; 3) Processing of mice embryos; 4) Storage of mice embryonic fibroblasts by freezing method and 5) Qualification of the MEF's by morphological, immunocytochemical and RT-PCR analysis.

The advantages of MEFs prepared in-house over the commercially available MEFS 3tt 3S
follows:
a) Readily available source of MEF.
b) No risk of loss of viability of the MEF cells by transportation.
c) The results of qualification of the indigenously prepared MEF is always available to verfy its efficiency in supporting growth of hES cells.
A population of cells derived, as described in the preferred embodiments express specific markers of embryonic stem cells like Oct-4, Nanog, Rexl, Sox-2, FGF4, Utfl, Thyl, Criptol, ABCG2, Dppa5, hTERT, Connexin 43, Connexin 45 and do not express markers characteristic of differentiated cells like Keratin 5, Keratin 15, Keratin 18, Sox-1, NFH (ectoderm), brachyury, Msxl, MyoD, HAND1, cardiac actin (mesoderm), GATA4, AFP, HNF-4alpha, HNF-3beta, albumin and PDX l(endoderm). The hES cells also express cell surface markers such as SSEA-
3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, E-coonexin 43 alkaline phosphatase as shown by immunocytochemistry. The extensive molecular characterization of the hES cell line of the present invention when compared to other commercially available cell lines may provide an edge for transplantation therapies.
The cells of the preferred embodiments exhibit high levels telomerase activity as assessed by non-radioactive PCR based SYBER green detection method. This is indicative of high self-renewal capacity of the cells of present invention for at least about 40 passages in culture, more preferably at least about 60 passages and most preferable at least 100 passages in culture. The hES cells also possesses normal euploid karyotype and show no gross alteration in the chromosomes even after one year in culture.
The present disclosure further describes an unique identification of the hES cells as evidenced by HLA and STR typing. HLA typing analyses plays a pivotal role as stem cell based transplantation therapies evolve. The exploitation of tandemly repeated elements in the genome by STR genotyping has become important in several fields including genetic mapping, linkage analysis, and human identity testing. The hES cell line as disclosed in the present invention

possess a unique HLA and STR type, which will provide a better match during transplantation for Indian population.
The human Eg cells in the present disclosure are pluripotent in nature which have the ability to
develop into representatives of all the three germ layers in vivo. When injected into SCID mice, human ES cell differentiates into cells derived from all three embryonic germ layers including: bone, cartilage, smooth muscle, striated muscle, and hematopoietic cells (mesoderm); liver, primitive gut and respiratory epithelium (endoderm); neurons, glial cells, hair follicles, and tooth buds (ectoderm). This was confirmed by examination of the histological sections of the tumor formed at the site of injection of the ES cells.
The derived hES cells are also capable of forming embryoid bodies (EBs) in suspension culture. The suspension aggregates were differentiated for 10-14 days in an ES medium without LIF. The EBs were shown to express a set of lineage specific markers including Keratin 5, Keratin 15, Keratin 18, Sox-1, NFH (ectoderm), Brachyury, Msxl, MyoD, HAND1, cardiac actin (mesoderm), GATA4, AFP, HNF-4alpha, HNF-3beta, albumin and PDX1 (endoderm). The unambiguous expression of a set of differentiated markers clearly demonstrates the differentiation potential of the hES cell line as disclosed in the present invention, wherein at least 80% of the differentiated cells were neurons, 30-50% were cardiomyocytes, 80-90% were hepatocytes, and 40-60% were pancreatic cells.
In other embodiments, the hES cells as described herein can be used to screen compounds, for example small molecules and drugs, for their effect on the cell population, The compounds can also be screened for cell toxicity or modulation.
In other embodiments, the hES cells according to the present disclosure can also be used to study the cellular and molecular biology of development, functional genomics, as well as the generation of differentiated cells for use in therapeutic or prophylactic transplantation, treatment, drug screening, or in vitro drug discovery. For example, the hES cells can be used for genomic analysis, to produce mRNA, cDNA, or genomic libraries, to produce specific polyclonal or monoclonal antibodies, including but not limited to humanized monoclonal antibodies (WO
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01/51616, specifically incorporated herein by reference), or to screen for the effects of different test compounds or biologically active molecules on hES cells and cells or tissues derived therefrom, such as pharmaceutical compounds in drug research. The test compounds or biologically active molecules screened may be derived for example from plants, plant-based
extracts, or synthetic sources. hES can be also be used to SCOT for factors (such as small
molecule drugs, peptides, polynucleotides, and the like) or conditions (such as cell culture conditions or manipulations) that affect the characteristics of hES cells in culture, and the differentiation of hES cells into various specific cell and tissue types.
To summarise, hES cells of the present disclosure are particularly advantageous due to several unique properties of these cells:
(1) hES cells are capable of differentiating into a variety of tissue types, belonging to all the three germ layers like endoderm, ectoderm, and mesoderm;
(2) hES cells are self-renewing and capable of propagating in culture for at least about 60 to about 100 passages or more while maintaining pluripotency, high telomerase activity, and normal karyotype;
(3) hES cells are capable of forming embryoid bodies (EBs).
(4) hES cells were maintained and propagated by manual passaging.
(5) hES cells can be used for treating various disorders in which cells degenerate or become dysfunctional including but not limited to neurological disorders, cardiac disorders, pancreatic disorders and hepatic diseases as disclosed in the present invention possess a unique HLA and STR type, which will provide a better match during transplantation for Indian population.
(6) hES cells can be used to screen compounds, for example small molecules and drugs, for their effect on the cell population, the compounds can also be screened for cell toxicity or modulation.
(7) hES cells can be used as an alternative to the conventional in vitro toxicity models for drug metabolism and toxicity studies, using the hES cell-derived hepatocytes, cardiomyocytes, neurons and pancreatic islet cells of the present invention.
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DETAILED DESCRD7TION OF THE PREFERRED EMBODIMENTS
Definitions: Pluripotent embryonic stem cells: As used herein the term "pluripotent human embryonic stem cells" refer to a cell that is derived from inner cell mass of blastocyst stage of mammalian embryo and are capable of self-regeneration and capable of differentiation to cells of all the three lineages such as ectoderm, mesoderm and endoderm. The pluripotent embryonic stem cells in the present invention are lineage uncommitted i.e. they are not committed to particular germ lineage such as ectoderm, mesoderm and endoderm. As used herein the term "Pluripotent stem cells" refer to cells that have high self-renewal capacity and possesses differentiation potential, both in vitro and in vivo. A pluripotent cell can be self-renewing and can remain dormant or quiescent within the tissue of organ.
Embryoid bodies: As used herein the term "embryoid bodies" refer to an aggregation of differentiated or undifferentiated pluripotent embryonic stem cells surrounded by primitive endoderm generated in suspension culture. Embryoid bodies contain cells of all three lineages including ectoderm, mesoderm and endoderm. Mouse embryonic stem cells studied since 20 years, also develop into 'embryoid bodies' containing cells characteristic of the three primitive layers of the embryo: endoderm, mesoderm, and ectoderm. In the embryo, each of these layers gives rise to cells of different phenotypes corresponding to each germ layer. In mature human embryoid bodies, it is possible to discern cells bearing markers of various cell types: neuronal cells, haematopoietic cells, liver cells, cardiac muscle cells and pancreatic islet cells. The! embryoid bodies and their detailed characterization can provide a valuable insight into the determination of the fate of embryonic stem cells. Further, we can modulate the differentiation of a desired phenotype through employment of suitable growth factors and their supplements.
Growth factors: As used herein the term "growth factor" refers to as proteins that bind to receptors on the cell surface with the primary result of activating cellular proliferation and differentiation through the activation of dormant signaling pathways. Majority of the growth factors/supplements are quite versatile, capable of stimulating cellular division in numerous different cell types. However, specificity of some of the growth factors are restricted to certain
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cell types. In the present invention growth factors used are specific to pluripotent embryonic stem cells and its induction to differentiation into various lineages such as neurons, hepatocytes, cardiomyocytes, beta-islets, chondrocytes, osteoblast, myocytes and the like.
Differentiation: As used herein the term "differentiation" refers to a process whereby undifferentiated embryonic stem cells acquire a state where cells are more specialized and has
characteristics of special tissues. These special tissues show the expression of tissue specific
markers at cellular and molecular level. The potential of differentiation of an embryonic stem cell line is the capacity of the said cell line to give rise to cell types belonging to all three germ layers like ectoderm, mesoderm and endoderm including teratocarcinoma. The in vitro differentiation potential of the ES cells can be demonstrated by culturing the cells under suitable conditions and the in vivo differentiation potential can be shown by injecting the cells into immuno-compromised (SCID) mice. Human embryonal carcinoma cells have a limited ability to differentiate into multiple cell types and represent the closest existing cell lines to ES cells.
Defining characteristics of human ES cells: Human embryonic stem cells share features with pluripotent human embryonal carcinoma cells (EC). Putative human ES cells may therefore be characterized by morphology and by the expression of cell surface markers characteristic of human EC cells. Additionally, putative human ES cells may be characterized by developmental potential, karyotype and immortality.
a) Morphology: The colony morphology of human embryonic stem cell lines is similar to, but distinct from, mouse embryonic stem cells. Both mouse and human ES cells have the characteristic features of undifferentiated stem cells, with high nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony formation. The colonies of human ES cells are flatter than mouse ES cell colonies and individual ES cells can be easily distinguished.
b) Cell surface markers: A human ES cell line of the present invention is distinct from mouse ES cell lines by the presence or absence of the cell surface markers described below. One set of glycolipid cell surface markers is known as the Stage-specific embryonic antigens 1 through 4. These antigens can be identified using antibodies for SSEA 1, preferably SSEA-3 and SSEA-4.
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NTERA-2 cl.Dl cell line was chosen for positive control only because it has been extensively studied and reported in the literature, but other human EC cell lines may be used as well.
Mouse ES cells (ES Jl) are used as a positive control for SSEA-1, and for a negative control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Other routine negative controls include omission of the primary or secondary antibody and substitution of a primary antibody with an unrelated specificity. Alkaline phosphatase may be detected following fixation of cells with 4% paraformaldehyde The globo-series glycolipids SSEA-3 and SSEA-4 are consistently present on
human EC cells. Differentiation of NTERA-2 CL Dl cells in vitro results in the loss of SSEA-3,
SSEA-4, TRA-1-60, and TRA-1-81 expression and the increased expression of the lacto-series glycolipid SSEA-1. This contrasts with undifferentiated mouse ES cells, which express SSEA-1, and neither SSEA-3 nor SSEA-4. Although the function of these antigens are unknown, their shared expression by Relicell™hESl cells and human EC cells suggests a close embryological similarity. Alkaline phosphatase will also be present on all human ES cells. A successful human ES cell culture of the present invention will correlate with the cell surface markers found in other
established human ES cell lines.
d) Developmental potential by teratoma formation: Human ES cells of the present invention are pluripotent. By "pluripotent" we mean that the cell has the ability to develop into any cell derived from the three main germ cell layers or an embryo itself. When injected into SCID mice, a successful human ES cell line will differentiate into cells derived from all three embryonic germ layers including: bone, cartilage, smooth muscle, striated muscle, and hematopoietic cells (mesoderm); liver, primitive gut and respiratory epithelium (endoderm); neurons, glial cells, hair follicles, and tooth buds (ectoderm).
d) Karyotype: Successful human ES cell lines have normal karyotypes. Both XX and XY cells lines can be derived. The normal karyotypes in human ES cell lines will be in contrast to the abnormal karyotype found in human embryonal carcinoma (EC), which are derived from spontaneously arising human germ cell tumors (teratocarcinomas). Although tumor-derived human embryonal carcinoma cell lines have some properties in common with embryonic stem cell lines, all human embryonal carcinoma cell lines derived to date are aneuploid. Thus, human ES cell lines and human EC cell lines can be distinguished by the normal karyotypes found in
17

human E5 cell lines and the atonal karyotypes found in human EC lines, By "normal
karyotype" it is meant that all chromosomes normally characteristic of the species are present and have not been noticeably altered. The normal karyotypes of the human ES cells suggests that this differentiation accurately recapitulates normal differentiation.
e) Immortality: Immortal cells are capable of continuous indefinite replication in vitro.
Continued proliferation for longer than one year of culture is a sufficient evidence for
immortality, as primary cell cultures without this property fail to continuously divide for this
length of time. Human ES cells will continue to proliferate in vitro with the culture conditions
described above for longer than one year, and will maintain the developmental potential to
contribute all three embryonic germ layers. This developmental potential can be demonstrated by
the injection of ES cells that have been cultured for a prolonged period (over a year) into SCID
mice and then histologically examining the resulting tumors. Although karyotypic changes can
occur randomly with prolonged culture, some human ES cells will maintain a normal karyotype
for longer than a year of continuous culture. This can be demonstrated by detection of the
telomerase enzyme activity of the human ES cells at the later stages of propagation. High levels
of telomerase activity are associated with cell proliferation during embryonic development and
with cell transformation and cancers.
f) Culture conditions: Growth factor requirements to prevent differentiation are different for the
human ES cell line of the present invention than the requirements for mouse ES cell lines.
Leukemia inhibitory factor (LIF) is necessary and sufficient to prevent differentiation of human
ES cells and to allow their continuous passage.
Alternatively, sources of human feeders including but not limited to human embryonic fibroblasts (HEF), human foreskin, bone marrow mesenchymal cells, stromal cells of various adult origin or any combinations thereof are being used in the present invention as an substitute to mouse embryonic feeders (MEF) to grow human ES cells with an objective to develop a xeno-free environment for human ES cell culture. Nevertheless, culture of human ES cells without feeders would be the ideal. Not only would this eliminate a possible source of exogenous contamination with potential pathogens, it would also greatly simplify the logistics of ES cell culture. For mouse ES cells, the discovery that LIF is able to support their self-renewal and
18

proliferation as undifferentiated cells in the absence of feeders was a significant advance. Unfortunately, LIF does not seem to have this ability with respect to human ES culture (Jones et al., 1998; Bongso et al., 2000). However, conditioned medium from mouse embryo fibroblasts will support the proliferation of human ES cells cultured on the extracellular matrix preparation Matrigel (Invitrogen) in the absence of feeders themselves (Carpenter et al., 2001). Although this provides some practical advantages, the active factor from the conditioned medium has not yet been identified, and this approach fails to eliminate the possibility of contamination with murine endogenous retroviruses.
g) Differentiation to extra embryonic tissues: When grown on embryonic fibroblasts and allowed to grow for two weeks after achieving confluence (i.e., continuously covering the culture surface), human ES cells of the present invention spontaneously differentiate into neurons, cardiomyocytes, hepatocytes and pancreatic islet cells. The markers responsible for the aforesaid cell types can be detected by semiquantitative RT-PCR and immunocytochemistry using gene specific primers and suitable antibodies respectively.
h) Differentiated stem cells in regenerative medicine: Human embryonic stem cells of the present invention may be induced to differentiate into particular phenotypes in vitro and a pure population of the desired cell type can be injected into damaged organ for the repair of injury. Such injury may be due to various disorders but not restricted to neuro-degenerative diseases, myocardial infarction, congestive heart failure, liver failure and diabetes. Neuro-degenerative diseases includes stroke, spinal cord injury, Parkinson's disease, Alzheimer's disease, multiple sclerosis and the like. Therefore, the differentiated hES cells possess enormous potential in cell transplantation for cell replacement therapy or tissue regeneration. The cell lines derived by the present invention can be used as a carrier vehicle for various therapeutically active molecules. The specific genes to be delivered at various sites of human body, preferably into the cells can be genetically manipulated as per the requirement and can be delivered to the target site for gene therapy.
i) Differentiated stem cells for drug screening and therapeutics: The present invention provides the possibility of using human embryonic stem cells and its unique capability to differentiate into the cells of all the three lineages such as ectoderm, mesoderm and endoderm for
19

pharmaceutical interventions and cell-based assays for drug discovery and in vitro toxicity testing. Another aspect of the present invention provides opportunity to use these differentiated cells not limited to neuronal cells, cardiomyocytes, hepatocytes and beta-islets to screen the
various biological active molecules pit in the plant-based extracts and synthetic sources. The
screening method can be used to develop novel drug molecules for various diseases preferably but not limited to Parkinson's diseases, Alzheimer's disease, Huntington disease, cardiac disorders, diabetes and hepatic diseases.
Cells of this invention can also be used to study xenobiotic-induced hepatotoxicity by measurement of the release of enzymes, including but not limited to serum elutamate pyruvate
amino-transferase (SGPT), serum glutamate oxalo-acetate aminotransferase (SGOT), alkaline
phosphatase (ALP) and lactate dehydrogenase (LDH). Further, hepatotoxicity and drug metabolism studies using hES cells of the present can be used to study drug-induced induction of cytochrome P450 isoforms, including but not limited to CYPIA1, CYP2A6, CYP2B6, CYP2C9, CYP2E1, CYP3A4 and to identify drug metabolite(s) using analytical techniques including (but not limited to^ HPLC, LC-MS, LC-MS/MS and GC-MS.
The cells derived from the present invention can also be used for generation of both polyclonal and monoclonal antibodies for either research or therapeutic potential, preferably for generating humanized monoclonal antibodies for the treatment of various diseases.
DETAILED DESCRIPTION OF THE INVENTION
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1
Preparation of blastocyst by in-vitro ferilization
Isolated blastocysts: Blastocyst stage embryos (blastocysts) may be Isolated from a variety of sources. These blastocysts may be isolated from recovered in vivo fertilized preimplantation embryos, or from in vitro fertilization (IVF), for example embryos fertilized by conventional insemination, intracytoplasmic sperm injection, or ooplasm transfer. Human blastocysts are obtained from couples or donors who voluntarily donate their surplus embryos. These embryos
are used for research purposes after acquiring written and voluntary consent from these couples
or donors. Alternatively, blastocysts may be derived by transfer of a somatic cell or cell nucleus into an enucleated oocyte of human or non-human origin, which is then stimulated to develop to the blastocyst stage. The blastocysts used may also have been cryopreserved, or result from embryos which were cryopreserved at an earlier stage and allowed to continue to develop into a blastocyst stage embryo. Preferably, blastocysts of good morphological grade are used in the present disclosure, for example blastocysts in which the inner cell mass is well developed. The
development of both the blastocyst and the inner cell mass win vary according to the species,
and are well known to those of skill in the art. The embryos are also cultured in medium conditions that maintain survival and enhance development into blastocyst stage embryos (Fong and Bongso, 1999, incorporated herein by reference).
Institutional Ethics Committee approval was obtained before initiation of any studies disclosed herein using human blastocysts. Prior written consent was taken from individual donors for the donation of surplus embryos for this study after completion of infertility treatments. The protocol generally used to obtain viable embryos from infertility patients is as follows:
In-vitro fertilization: For IVF, the woman needs to undergo pituitary suppression or down regulation with GnRH agonist [injection Leuprolein Acetate (Lupron)]. This is followed by controlled ovarian hyperstimulation with injection of Gonadotrophin (hMG) for 7-12 days which is monitored for growth of the follicles by ultrasonography and plasma estradiol levels.

Ovulation is triggered by intramuscular injection of hCG 10.000 IU (Profasi) when at least one or more follicles are 18 mm in diameter.
Oocyte retrieval and recovery of embryos: Oocytes retrieval is achieved by follicular aspiration 34-36 hours under ultrasonography guidance. Fertilization is assessed by the presence
of 1 pronuclei (2 PN) and the fertilized oocytes are transferred to embryo culture dish. Two fertilized oocytes (2 PN) per plate are transferred in 0.75 -1 ml of cleavage medium [Quinn's
cleavage Medium (Sage Biopharma Cat. # ART-1026). These dishes are incubated in the incubator in a 5% CO2 environment at 37°C until day 2. On day 2, the cleavage medium is changed. On day 3, blastocyst medium [QA BLastocytes Medium (Sage Biopharma Cat. # ART-1029)] replaces the cleavage medium and the embryos are cultured until day 5 to day 7 until expanded blastocysts are obtained. Medium is replaced every alternate day. After overnight culture the embryos were monitored visually under a dissecting microscope. The integration was considered successful if the embryo developed into a morula or well expanded blastocyst (Fig 1.1 and 1.2). The present invention can be carried out from the morula stage to the blastocyst stage.
EXAMPLE 2
Derivation and storage of mouse embryonic fibroblast (feeder) cells
Procurement of pregnant mice and dissection: Mouse embryonic fibroblasts (MEFs) were obtained from inbred C57 Black mice or suitable strains. In an illustrative method, a mouse at 13.5 days of pregnancy/days post coitum (dpc) is sacrificed by cervical dislocation. The abdomen of the mouse was swabbed with 70% Isopropanol followed by a small incision. The viscera was exposed by pulling apart the abdominal skin in opposite directions. The uteri filled with embryos was seen in the posterior abdominal cavity. The uterus was dissected out with sterile forceps and scissors and placed into 50 ml screw capped conical centrifuge tube containing 20 ml of sterile Dulbecco's phosphate buffered saline, Ca and Mg free (Gibco-BRL,

the help of sterile pointed forceps and scissors and then placenta, membrane and soft tissues were removed.
Staging of mice embryos: Mouse embryos were staged under the dissecting microscope. Staging of the mouse embryos can be done according to a variety of criteria, the most general of which are described by Theiler in " The House Mouse: Atlas for mouse development" (1989). Theiler's criteria are too broad to distinguish many important phases of early development and must therefore be supplemented by others, for example, cell number, somite number or those
characteristics used by Downs and device (1993), same gestation age may differ in their stage
of development. The stages recognized by Downs and Davis is applicable to Fl hybrid of C57 Black X CBA mice, inbred C57 black mice and other closely related strains. The most acceptable stages for obtaining feeders for the purpose of growing human embryonic stem cells is Theiler stage 21 and 22. Theiler stage 21 is 13dpc with a range of 12.5-14 and 52-55 somite stage. This stage is identified as an anterior foot-plate indented, elbow and wrist identifiable, 5
OWS Of whiskers and umbilical hernia clearly apparent. Hair follicles are absent and fingers are
distally separate. Theiler stage 11 is recognised as 14dpc with range of 13.5 to 15, 56- 60 somite stage. The distinguishing features of this stage are fingers separate distally, only indentation between digits of the posterior foot-plate, long bones of limbs present, hair follicles in pectoral, pelvic and trunk regions are present. Other features include absence of open eyelids, hair follicles in cephalic regions.
Processing of mice embryos: The embryos were further processed by first discarding the head followed by all visceral organs under the dissecting microscope with the help of sterile pointed forceps. The carcass was then transferred into the lid of a 96 mm sterile petridish and minced properly with the help of sterile curved scissors. The minced mass is then transferred into 50 ml conical centrifuge tube containing approximately 15-20 ml of 0.25% Trypsin-EDTA (Gibco-BRL, Catalog No. 25200-056), pre-warmed at 37°C. The minced mass was then titurated 3-4 times in the Trypsin-EDTA solution with the help of a 10 ml pipette and passed 2-3 times though a 20 ml syringe fitted to a 18 gauge needle. The cell suspension was then incubated for 10-15 minutes at 37°C. The cell suspension was once again titurated through a 10 ml pipette. The trypsin in the cell suspension was inactivated by adding 20 ml of complete media (90% Dullbecco's modified Eagle's medium- High Glucose, 10% Fetal bovine serum, 1 mM L-

Glutamine, 1% Non-Essential amino acids and 0.1 mM P- Mercaptoethanol) and the cell suspension was finally plated in tissue-culture flask. Thereafter, the cells were grown till confluency with media change every alternate day with periodic monitoring.
Freezing of mouse embryonic fibroblasts: Freezing of the cells was done at confluency in freezing media comprising of 60% Fetal bovine serum, 20% DMSO and 20% complete media. For freezing, the cells were resuspended in complete media and then mixed with freezing media in the ratio 1:1. This freezing suspension was then dispensed 1 ml each in cryovials such that one ml contains 5 million cells.. These vials were then stored in liquid nitrogen for long term use.
Qualification of MEFs: Every batch of feeders are qualified after growing the hES cells for 5 passages, on the respective feeder layers (MEF) to be qualified. The process of qualification involves assessment of critical parameters like morphological analysis of the hESC colonies (Fig 2.1 & 2.2), expression of ES cell markers by imuunochemistry (Fig 2.3 & 2.4), RT-PCR (Fig 2.5) and steriliy check by endotoxin and mycoplasma testing. Only qualified feeders were used for isolation, passaging and maintenance of Relicell™hESl.
EXAMPLE 3
Derivation and maintenance of human ES cells
Inactivation and plating of mouse embryonic fibroblast (feeder) cells: The feeder cells stored in liquid nitrogen were revived as per need. The vials were thawed by plunging the frozen vials in 37°C water bath till the contents are semi thawed. The contents were then collected in a tube and mixed with warm media to dilute the cryoprotectant. The cells were then pelleted down and plated in fresh MEF media {90% Dullbecco's modified Eagle's medium- High Glucose (Gibco), 10% Fetal bovine serum (Hyclone), 1 mM L-Glutamine (Gibco), 1% Non-Essential amino acids (Gibco) and 0.1 mM P- Mercaptoethanol (Sigma)} in tissue culture flasks. Once the cells reach confluence, they are ready for inactivation. The cells were inactivated by Mitomycin C treatment or by gamma irradiation. In the given invention, the cells were inactivated by Mitomycin C treatment for two and half hours. 10ng/ml of Mitomycin C was used for inactivation at 37°C and 5% C02. The cells were then washed several times for complete removal of Mitomycin C and the trypsinised using enzymes like trypsin-EDTA. These cells were then counted and plated onto
24

0.2% gelatinized plates at a concentration of cells/cm2. The cells were plated and incubated at 37°C and WoCo2.Tnese plates were then used for plating of hES cells.
ICM isolation: To isolate ICM without risking cell loss, whole embryo culture method was employed on Day 6 of the embryo culture (Fig 1 and Fig 3). The zona-pellucida was digested by 0.5% pronase for about 2 minutes. The ICM was then plated on mitotically inactivated mouse embryonic fibroblast (MEF) cells. The hES culture medium used in this technique consists of
80% DMEM/F-12 (Gibco, with glucose 4500mg/L), 15% ES tested FBS (Hydone, USA), 5%
Serum replacement (Gibco, #10828-028), 1% nonessential amino acid solution (Gibco), ImM
glutamine (Gibco), 0.1% beta mercaptoethanol (Sigma), 4ng/ml human bFGF (R&D systems) and 10ng/ml human Leukemia inhibitory factor (Sigma). After 7 days, ICM clump was separated from other cells by mechanical dissociation with a micropipette .The ICM clump was then replated on fresh feeder cell layer and fresh medium was added.
Culturing and manual passaging of hES cells; Subsequent passaging of the undifferentiated
colonies was done by cutting the colonies systematically in clumps of about 100 cells using the sharp edge of a glass-pulled micropipette (Fig 4). Selection was done to remove any unwanted differentiated areas of the colony. As soon as the clumps detached they were picked up by the same micropipette (with bore size slightly bigger than the size of the clump) attached with a mouth aspiration set and transferred to a fresh fibroblast feeder layer. The culture system is maintained at a constant temperature of 37 degree C by placing it in a 5% C02 incubator. The cell line Relicell™hEl has been grown for 40 passages in vitro and the cell line still consist mainly of cells with the morphology of ES cells.
Cryopreservation of hES cells: Three days old 'good' undifferentiated human ES colonies were used for freezing. ES colonies along with the feeder layer were cut into small pieces using a cell scrapper. Then the cells were collected in a sterile 15ml centrifuge tube (Nunc) and spun at 200G for 3 minutes. The supernatant was aspirated out. The volume of the cell pellet was measured and resuspended in hES media to make up the volume to 0.5 ml. Equal volume of freezing medium which included 60% ES tested FBS (Hyclone, USA), 20% hES medium, 20% DMSO HYBRIMAX (Sigma) was added gently to the hES cell suspension with occasional swirling. Clumps of ES cells were transferred into a 1.2 ml cryo-vial (Nalge-Nunc, USA)

containing freezing medium. The vials were slowly cooled (~1 c/min) in a freezing container (Sigma) to -80 c and next day stored in liquid nitrogen. On revival, post thaw survivability of the frozen hES cells was found to be about 50% or more.
EXAMPLE 4: Characterization of human ES cells
Generation of embryoid bodies: The hES colonies need to be either cut into small pieces manually or dissociated into small pieces by enzymatic treatment with collagenase or trypsin EDTA. In the given invention, the hES colonies were cut manually into small pieces for embryoid body formation. The small pieces were then transferred to bacteriological plates for aggregation in EB medium {80% DMEM/F-12 (Gibco, with glucose 4500mg/L), 15% ES tested FBS (Hyclone, USA), 5% Serum replacement (Gibco, #10828-028), 1% nonessential amino acid solution (Gibco), ImM glutamine (Gibco), 0.1% beta mercaptoethanol (Sigma)}. The cell aggregates were allowed to grow in this medium for 10-14 days with media change every three days. The embryoid bodies generated by this method were characterized for cellular and molecular markers at different days in suspension cultures like Od, 6d, 10d and 14d (Fig 8.1 to 8.4) to evaluate the in vitro differentiation potential of the hES cell line.
Immunocytochemistry: The cells grown in 2-well chamber slides (Becton Dickinson, USA) were fixed in freshly prepared 4% paraformaldehyde and permeabilized with 0.2 % Triton X-100 in PBS. The non-specific binding sites were blocked with 1% bovine serum albumin in PBS. The cells were then incubated overnight at 4°C with primary antibody. Here, we have checked a panel of undifferentiated stem cell markers like Oct-3/4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, alkaline phosphatase, Connexin 43, E-cadherin (Fig 5.1 to 5.8) and a group of differentiated markers like nestin (Ectoderm), smooth muscle actin, brachyury (Mesoderm), AFP, GATA4 (Endoderm) (Fig 9.1 to 9.5) (Please refer Table I for details of Antibodies). Cells were then washed and incubated with the appropriate FITC labeled secondary antibody at room temperature for 1 hour in the dark. Cells were counterstained with DAPI (lug/ml; Sigma). After mounting, the cells were observed under a fluorescence microscope (Nikon Eclipse E600) to evaluate immunopositive areas. Human embryonic stem cells expressed the globo-series cell surface antigens like SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and the POU5fl encoded transcription factor, Oct-4 typical of human ES cells along with E-cadherin and Connexin-43
26

(Fig 5). The human ES cells also exhibited alkaline phosphatase activity as evidenced by fluorescence microscopy (Tig 5.8). Further, the 14d embryoid bodies stained positive for
differentiation markers like nestin (ectoderm), smooth muscle actin, brachyury (mesoderm) and GATA4, AFP (endoderm) (Fig 9).
Table I: Details of antibodies used

Name of the antibody Manufacturer Dilution used
Oct-3/4 Santacruz, USA 1:100
SSEA-1 ES cell characterization kit(Chemicon; Cat # SCR001) 1:40
SSEA-3
SSEA-4
TRA-1-60
TRA-1-81
Alkaline phosphatase
E-cadherin Santa, USA 1200
Connexin 43 Santacruz, USA 1:200
Nestin Chemicon, USA 1:200
Smooth muscle actin Santacruz, USA 1:100
GATA4 Santacruz, USA 1:100
Gene expression analysis by RT-PCR: Total RNA was isolated by TRIzol method (Invitrogen) according to the manufacturer's protocol. 1 ug of RNA treated with RNase-OUT ribonuclease inhibitor (Invitrogen) was used for cDNA synthesis. Reverse-transcription using Superscript reverse transcriptase-II (Invitrogen) and Oligo dT (Invitrogen), to prime the reaction was carried out. PCR primers were selected to distinguish between cDNA and genomic DNA by using individual primers specific for different exons. 1 u.1 of cDNA was amplified by polymerase chain reaction using Abgene 2X PCR master mix (Abgene, Surrey, UK) and appropriate primers. The expression of an array of undifferentiated stem cell markers like Oct-4, Nanog, Rexl, Sox-2, FGF4, Utfl, Thyl, Criptol, ABCG2, Dppa5, TERT, Connexin 43, Connexin 45; lineage specific markers including Keratin 5, Keratin 15, Keratin 18, Sox-1, NFH (ectoderm), Brachyury, Msxl, MyoD, HANDl, cardiac actin (mesoderm), GATA4, AFP, HNF-4alpha, HNF-3beta, albumin and PDX1 (endoderm) were checked (please refer Table II for details of primers). For all the

genes, PCR were performed for 35 cycles, consisting of an initial denaturation at 94° C for 1 min, then 94° C for 30 sec, annealing temperature of the respective gene primer for 45 sec (for Tm values of individual primers please refer Table II), 72° C for 1 min and was terminated by final extension at 72° C for 5 mins. The hES cells at early as well as late passages exhibited an unambiguous expression of a set of genes associated with pluripotency including Oct-4, Nanog,
Ik-1, Sox-2, Cfiptol, FGF4, Tkyl, Utfl, ABCG2, Dppa5, kTERT out also the gap junction
proteins like Connexin 43 and Connexin 45 (Fig. 6 and Table III). HEF cells, used as a negative control was devoid of the expression of any of these markers. Further, the expression profile of an exhaustive list of genes related to lineage specific differentiation was evaluated with Od, 6d, 10d and 14d old embryoid bodies (Fig. 10 and Table III). A consistent expression of early stage ectodermal markers like Keratin 5, Keratin 15 and Keratin 18 from 6d to 14d of differentiation
w observed, with no expression on the 0th day of differentiation, although the late stage
neviroectodermal markers Sox-1 and NFH were present only on the 10th to 14 day of differentiation (Fig 10). Among the mesodermal lineage markers, Msxl, a pre-cardiac transcription factor was seen to be expressed uniformly throughout the progressive days of differentiation. But, the other mesodermal markers including brachyury, HAND1, MyoD and cardiac-actin demonstrated weak or no expression in the hES cells (Fig 10). Similarly, the early endodermal cell markers including AFP, HNF-4alfa and HNF-3beta exhibited an expression on
the 6th and 10th day of cell aggregate formation while GATA4 demonstrated a transient increase
from 10 day upto 14 day of suspension culture (Fig 10). However, very weak expressions were detected with the markers for mature hepatocytes and pancreatic islet cells, albumin and PDX1 respectively, thereby indicating absence of mature endodermal derivatives (Fig 10).
Table II: Details of primers used

Gene Primer sequence Annealing temp Expected
(degC) Product size (bp)
Housekeeping gene
GAPDH 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'5 '-C ATGTGGGCCATGAGGTCCACCAC- 60 892

3'
28

Pluripotent stem cell markers
Oct-4 5'-CGRGMGCTGGAGAAGGAGAAGCTG-3'5 '-CAAGGGCCGCAGCTTACACATGTTC-3' 58 247
Nanog 5 '-CCTCCTCCATGGATCTGCTTATTC A-3'5'-CAGGTCTTCACCTGTTTGTAGCTGAG-3' 52 262
Rexl 5 '-GCGTACGC AAATTAAAGTCCAGA-3' 5'-CAGCATCCTAAACAGCTCGCAGAAT-3' 56 306
Sox2 5'- CCCCCGGCGGCAATAGCA -3' 5'- TCGGCGCCGGGGAGATACAT-3' 55 448
Thyl 5' CATGAGAATACCAGCAGTTCACCCA-3'5' CACTTGACCAGTTTGTCTCTGAGCA-3' 55 272
FGF4 5" - CTACAACGCCTACGAGTCCTACA-3'5 '-GTTGC ACC AG AAAAGTC AGAGTTG-3' 53 370
ABCG2 5'-GTTTATCCGTGGTGTGTCTGG-3' 5 '-CTGAGCTATAGAGGCCTGGG-3' 62 684
Dppa5 5'-ATGGGAACTCTCCCGGCACG-3' 5 '-TCACTTCATCC AAGGGCCTA-3' 62 353
Utfl 5'-ACC AGCTGCTGACCTTG AAC-3' 5 '-TTGAACGTACCC AAGAACGA-3' 60 230
Criptol 5 '-AC AG AACCTGCTGCCTG AAT-3' 62 217
29

5 '-ATC ACAGCCGGGTAGAAATG-3'
hTERT 5'-AGCTATGCCCGGACCTCTAT -3' 5 '-GCCTGCAGCAGGAGGATCTT-3' 60 165
Gap Junction Proteins
Connexin 43 5 '-TACCATGCGACC AGTGGTGCGCT-3' 5 '-GAATTCTGGTTATCATCGGGGAA-3' 64 295
Connexin 45 5 '-CTATGCAATGCGCTGGAAACAACA-3'5'-CCCTGATTTGCTACTGGCAGT-3' 64 819
Ectodermal markers
Keratin 8 5 '-TGAGGTC AAGGC AC AGTACG-3' 5 '-TGATGTTCCGGTTCATCTCA-3' 60 161
Keratin 15 5 '-CACAGTCTGCTGAGGTTGGA-3' 5 '-GAGCTGCTCC ATCTGTAGGG-3' 62 196
Keratin 18 5'-GGAGGTGGAAGCCGAAGTAT-3' 5'-GAGAGGAGACCACCATCGCC-3' 60 164
Sox-1 5'-TACAGCCCCATCTCCAACTC-3' 5 '-GCTCCGACTTC ACC AGAGAG-3' 60 201
NFH 5 '-TGAACACAGACGCTATGCGCTCAG-3'5'-C ACCTTTATGTGAGTGGAC AC AGAG-3' 58 400
Mesodermal markers
Brachyury 5 '-TAAGGTGGATCTTCAGGTAGC-3' 5 '-CATCTCATTGGTGAGCTCCCT-3' 60 251
MyoD 5 '-GTCGAGCCTAG ACTGCCTGT-3' 5 '-GGTATATCGGGTTGGGGTTC-3' 60 217
Msxl 5 '-CCTTCCCTTTAACCCTCAC AC-3' 5 '-CCGATTTCTCTGCGCTTTTC-3' 62 287
HAND1 5 '-GCCTAGCCACCACTGCGCTTTTC-3' 5 '-CGGCTC ACTGGTTTAACTCC-3' 62 389
30

Cardiac-Actin 5 '-TCTATGAGGGCTACGCTTTG-3' 5' -CCTGACTGGAAGGTAGATGG-3' 50 630
Endodermal markers
AFP 5'-AGAACCTGTCACAAGCTGTG-3' 5 '-GAC AGC AAGCTGAGGATGTC-3' 62 577
GATA4 5 '-CTCCTTCAGGC AGTGAGAGC-3' 5 '-GAGATGC AGTGTGCTCGTGC-3' 52 680
HNF-4alfa 5'-TCTCATGTTGAAGCCACTGC-3' 5 '-GGTTTGTTTCTCGGGTTGA-3' 50 501
HNF-3beta 5 '-GAC AAGTGAGAGAGC AAGTG-3' 5 '-ACAGTAGTGGAAACCGGAG-3' 56 237
Albumin 5'-CCTTTGGCACAATGAAGTGGGTAACC-3'5 '-CAGCAGTCAGCCATTTCACC ATAGG-3' 58 450
PDX1 5 '-GTCCTGGAGGAGCCCAAC-3' 5 '-GCAGTCCTGCTCAGGCTC-3' 62 362
Table III: Summary of gene expression analysis

Serial Number Name of the gene Observed expression
ReliceUlMhESl BG01 hESC line(Brimble et. al, 2004) HEF
Housekeeping gene
1. GAPDH + + +
Pluripotent stem cell markers
2. Oct-3/4 + + -
3. Nanog + + -
4. Rexl + + -
5. TDGF1 + + -
31

6. Thyl + NR +
7. Sox-2 + + -
8. FGF4 + NR -
% Utfl + + -
10. ABCG2 + + -
11. Dppa5 + + +
12. Cripto + + -
13. TERT + + -
Gap junction proteins
14. Connexin 43 + + -
15. Connexin 45 + + -
Ectodermal markers in ce 1 aggregates
16. Keratin 8 + + -
17. Keratin 15 + + -
18. Keratin 18 + + -
19. NFH + + -
20. Sox-1 + + -
Mesodermal markers in cell aggregates
21. Brachyury + + -
22. MyoD + + -
23. Msxl + + +
24. HAND1 + + -
25. C-actin - + -
Endodermal markers in ce 1 aggregates
26. GATA4 + + -
27. AFP + + -
28. HNF4a - NR -
29. HNF3b + + -
30. Albumin - NR -
31. PDX1 + + -

HLA typing: Genomic DNA was isolated from the hES cells grown using the Qiagen DNA
isolation Kit, HLA DNA typing was performed by utilizing an adopted hybridization of PCR
amplified DNA with sequence specific primers (SSP) as the primary technology for HLA typing. The Oleorup SSP™ HLA-A-B-DR Combi tray kit (low-resolution method) for this experiment was used. In summary, after DNA isolation, amplification of the alleles for HLA-A-B-DR was carried out by employing low-resolution primer sets with well defined negative controls on 96 well plate. Assays were performed to analyze the HLA-A, HLA-B and HLA-DRB loci. Data
was interprted with the help of Genovision software,
Table IV: HLA profile

Test Sample HLA-A HLA-B HLA-DRB1 HLA-DRB3
Relicell1MhESl Allele 1 Allele2 Allele 1 Allele 2 Allele 1 Allele 2 Absent
01 02 0733 35 01
35 35 01 01
35 56
35 8301
STR Typing: Frozen Relicell hESl cells grown on MEF feeders were resuspended in IX PBS (Phosphate Buffered Saline). A 20mL aliquot was spotted on a labeled FTA card (Whatman) and allowed to dry. The FTA card lyses the cells on contact and binds the DNA to the paper surface. Prior to PCR, a portion of the dried spot was removed with a Harris punch, washed three times with Purification Reagent (Whatman), washed once with TE Buffer (Tris-EDTA pH 8.0), and allowed to dry. STR analysis was conducted using the multiplex-PCR-based PowerPlex 1.2 kit (Promega). Loci analyzed include D5S818, D13S317, D7S820, D16S539, vWA, TH01, Amelogenin, TP0X and CSF1P0. Electropherogram data were collected on an ABI 310 Genetic Analyzer. Data was analyzed using Genescan 3.1 and Genotyper 2.0 (Applied Biosystems).

Karyotype: Karyotyping was performed using standard methods of colcemid arrest and G-banding technique. Briefly, hES cells cultured in a 60 mm culture dishes till 60% confluency. The cells were incubated with ethidium bromide (12ug/ml) for 40 mins at 37°C, 5% C02 followed by colcemid (120ng/mL) treatment for 40 mins. The cells were dissociated with pre-warmed 0.25% trypsin-EDTA. The cells were then collected by centrifugation, resuspended in
hypotonic KC1 solution (0,075 M) for 15 minutes, and then fixed in Carnoy's fixative (glacial
acetic acid: methanol; 3:1). Metaphase spreads were prepared on wet glass microscope slides, air dried, baked at 90° C for an hour and Giemsa staining was performed. 20 metaphases were fully karyotyped using an Olympus BX40 microscope and images were captured using the Cytovision digital imaging system.
Telomerase assay: Telomerase assay was performed using non-radioisotopic gel based standard TRAP (Telomerase Repeat Amplification) protocol (Gang et al.2000 and Rubiano et al. 2003) using TRAPeze telomerase detection kit by Chemicon, USA (Catalog No.S7700). Approximately, 50-70 colonies of the human embryonic stem cells were pelleted and lysed using 200 ul of IX CHAPS lysis buffer. The cell suspension in IX CHAPS lysis buffer was incubated in ice for 30 minutes and then centrifuged for 20 minutes at 12,000g at 4 °C. The supernantant was quickly frozen and stored at -80°C. The total protein is estimated using Bradford assay. Telomerase assay is performed using 1- 6jxg of total extract. Heat inactivated samples served as the respective negative controls for each assay. For telomerase PCR, the master mix was prepared by adding dNTP, TRAP Primer mix, TS primer and TAQ polymerase as per the amounts mentioned in the kit. Finally^ the cell extract was added and the total reaction volume was maintained to 50ul. A two-step PCR reaction was performed (94°C for 30 seconds and 59°C for 30 seconds) for 33 to 35 cycles. The PCR products were electrophoresed on 12.5% Non- denaturing Polyacrylamide vertical gel at 400 volts till the xylene-cyanol dye front reaches two thirds of the entire runlength. The gel was then stained with 1:5000 dilution of SYBR GREEN I dye (Molecular Probes, Catalog No.S-7567), visualized under UV transilluminator and photographed using a gel documentation system. The relative quantitation of the telomerase product generated (TPG) is done as per the method of Gang et al. 2003. The TPB is explained by the formula: TPG = { [ (TP-B) / T I] / [(R8- B) / RI] }. Here, TP - Telomerase product generated in test extract; B - Telomerase product generated in Blank lysis buffer; R8 -Telomerase product generated in Quantification standard, TSR8 control template; TI - Internal
34

control of test extract; RI - Internal control of quantification standard, T5R5 control template,
Figure 7 shows high telomerase activity of ReliCell^~hESl at passage 37, with NTERA-2 hEC cells as a positive control and MEF as the negative control.
Sterility and Pathogen Testing: Extensive bacterial and fungal tests were performed on the Relicell™hESl cell cultures. The cultures were routinely monitored and reported at 48 hour, 14
day aid 21 day nitons, Additionally, endotoxin and mycoplasma testing were performed
using a Hoechst Assay for each culture. Finally, the cultures were screened for the presence or human pathogens including HIV-1, HIV-2, Human T-Cell Lymphotrophic Virus I/II, HSV1, HSV2, EBV, CMV, Hepatitis B Virus and Hepatitis C Virus.
Teratoma formation: Adult NUDE mice were used for teratoma formation study. Undifferentiated human embryonic stem cell suspension (5.0-10 million cells per animal) was
injected intramuscularly, After injection, the animal was kept in an individual filter top cage,
These cages were housed in special animal isolators to prevent any possible infection. After 8-10 weeks, the animals were sacrificed with an overdose of Ketamine (100 mg/kg i.p.) and was perfused transcardially with heparin saline (0.1 heparin in 0.9 % saline) followed by 4 % paraformaldehyde prepared in phosphate buffered saline. The tumor was dissected out and was fixed overnight in 4% paraformaldehyde along with 20 % sucrose. The tumor was sectioned (20 um) using cyro-microtome and sections were collected on the gelatin-coated slides. The tumor sections were stained with Hematoxylin/ Eosin and observed under the microscope for cells belonging to three germ layers including ectoderm, mesoderm and endoderm. All animal experiments were carried out following the guidelines of Institutional animal ethics committee.
All of the compositions and methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and

modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.
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Dated this 17 th day of May 2005.
For RELIANCE LIFE SCIENCES PVT.LTD..

K.V.SUBRAMANIAM
Sr. Executive Vice President
To:
The Controller of Patents
The Patent Office
Mumbai.
39

ABSTRACT
A purified preparation of human embryonic stem cells is disclosed. This preparation is characterized by the positive expression of the following pluripotent cell surface markers: SSEA-1 (-); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); alkaline phosphatase (+) and a set of ES cell markers including Oct-4, Nanog, Rexl, Sox2, Thyl, FGF4, ABCG2, Dppa5, UTF1, Criptol, hTERT, Connexin-43 and Connexin-45. The cells of the preparation are negative for lineage
specific markers like Keratin 8, Sox-1, NFH (ectoderm), MyoD, brachyury, cardiac-actin
(mesoderm) and HNF-3 beta, albumin, PDX1 (endoderm). The cells of the preparation are human embryonic stem cells, have normal karyotypes, exhibit high telomerase activity and continue to proliferate in an undifferentiated state after continuous culture for over 40 passages. The embryonic stem cell line Relicell™ hESl also retain the ability, throughout the culture, to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a human embryonic stem cell line is also disclosed.
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