Patent Application: US-50011804-A

Abstract:
the present invention concerns a method for the establishment of pluripotent human blastocyst - derived stem cell lines , stem cells obtained by the method , differentiation of these cells into differentiated cells , the differentiated cells , and the use of these differentiated cells in the preparation of medicaments . the undifferentiated pluripotent stem cells can be made to differentiate to a number of specialized cell types which can be utilized in the manufacture of medicaments for treating a number of conditions or pathologies involving degeneration of tissue , e . g ., of the pancreas leading to , e . g ., development of diabetes , or of the cns or degeneration of the cns caused by e . g ., stroke or physical trauma .

Description:
the inventors have established a novel method for establishing a pluripotent human blastocyst - derived stem cell line from a fertilized oocyte , including propagation of the cell line in an undifferentiated state . thus , the present invention relates to a method for obtaining a pluripotent human blastocyst - derived stem cell line , the method comprising the steps of i ) using a fertilized oocyte optionally , having a grade 1 or 2 , to obtain a blastocyst , optionally having a grade a or b , ii ) co - culturing the blastocyst with feeder cells for establishing one or more colonies of inner cell mass cells , iii ) isolating the inner cell mass cells by mechanical dissection , iv ) co - culturing of the inner cell mass cells with feeder cells to obtain a blastocyst - derived stem cell line . v ) optionally , propagation of the blastocyst - derived stem cell line . in accordance with to the above , it is one object of the present invention to provide a method for establishing an undifferentiated human blastocyst - derived stem cell line . as a starting material for this procedure , fertilized oocytes are used . the quality of the fertilized oocytes is of importance for the quality of the resulting blastocysts . in the method of the present invention , the establishment and evaluation of blastocysts are performed as described below . the human blastocysts in step i ) of the method may be derived from frozen or fresh human in vitro fertilized oocytes . in the following is described a procedure for selecting suitable oocytes for use in a method according to the present invention . the present inventors have found that an important success criterion for the present method is a proper selection of oocytes . thus , if only grade 3 oocytes are applied , the probability of obtaining a hbs cell line fulfilling the general requirements ( described below ) is low . donated fresh fertilized oocytes : on day 0 the oocyte is aspirated in asp - 100 ( vitrolife ), and fertilized on day 1 in ivf - 50 ( vitrolife ). the fertilized oocyte is evaluated based on morphology and cell division on day 3 . the following scale is used for fertilized oocyte evaluation : grade 1 fertilized oocyte : even blastomers , no fragments grade 2 fertilized oocyte : & lt ; 20 % fragments grade 3 fertilized oocyte : & gt ; 20 % fragments after evaluation on day 3 , fertilized oocytes of grade 1 and 2 are either implanted or frozen for storage . fertilized oocytes of grade 3 are transferred to icm - 2 ( vitrolife ). the fertilized oocytes are further cultured for 3 - 5 days ( i . e . day 5 - 7 after fertilization ). the blastocysts are evaluated according to the following scale : grade a blastocyst : expanded with distinct inner cell mass ( icm ) on day 6 grade b blastocyst : not expanded but otherwise like grade a grade c blastocyst : no visible icm donated frozen fertilized oocytes : at day 2 ( after fertilization ) the fertilized oocytes are frozen at the 4 - cell stadium using freeze - kit ( vitrolife ). frozen fertilized oocytes are stored in liquid nitrogen . informed consent is obtained from the donors before the 5 - year limit has passed . the fertilized oocytes are thawed using thaw - kit ( vitrolife ), and the procedure described above is followed from day 2 . as described above , fresh fertilized oocytes are from grade 3 quality , and frozen fertilized oocytes are from grade 1 and 2 . according to data obtained by the methods of the present invention , the percentage of fresh fertilized oocytes that develop into blastocysts is 19 %, while 50 % of the frozed fertilized oocytes develop into blastocysts . this means that the frozen fertilized oocytes are much better for obtaining blastocysts , probably due to the higher quality of the fertilized oocytes . 11 % of the blastocysts derived from fresh fertilized oocytes develop into a stem cell line , while 15 % of the blastocysts derived from frozen fertilized oocytes develop into a stem cell line . in summary , of the fertilized oocytes that were put into culture 2 % of fresh fertilized oocytes developed into a stem cell line , and 7 % of frozen fertilized oocytes that were put into culture developed into a stem cell line . the culturing of the fertilized oocyte to the blastocyst - stage is performed after procedures well - known in the art . procedures for preparing blastocysts may be found in gardner et al , embryo culture systems , in trounson , a . o ., and gardner , d . k . ( eds ), handbook of in vitro fertilization , second edition . crc press , boca raton , pp . 205 - 264 ; gardner et al , fertil steril , 74 , suppl 3 , o - 086 ; gardner et al , hum reprod , 13 , 3434 , 3440 ; gardner et al , j reprod immunol , in press ; and hooper et al , biol reprod , 62 , suppl 1 , 249 . after establishment of blastocysts in step i ) optionally derived from fertilized oocytes having grade 1 or 2 , the blastocysts having grade a or b are co - cultured with feeder cells for establishing one or more colonies of inner cell mass cells . after being plated onto feeder cells , their growth is monitored and when the colony is large enough for manual passaging ( approximately 1 - 2 weeks after plating ), the cells may be dissected from other cell types and expanded by growth on new feeder cells . the isolation of the inner cell mass cells is performed by mechanical dissection , which may be performed by using glass capillaries as a cutting tool . the detection of the inner cell mass cells is easily performed visually by microscopy and , according , it is not necessary to use any treatment of the oocytes with enzymes and / or antibodies to impair or remove the trophectoderm . thus , the procedure alleviates the need for immunosurgery . by comparing the success - rate in using immunosurgery versus the present method , which leaves the trophectoderm intact , it has been observed that the much simpler , faster and non - traumatic procedure of avoiding immunosurgery is more efficient than immunosurgery . the novel procedures make the preparation of stem cell lines , and the differentiation of these cell lines commercially feasible . from a total of 122 blastocysts , 19 cell lines were established ( 15 . 5 %). 42 blastocysts were processed by immunosurgery and 6 of these resulted in successfully established cell lines ( 14 %). eighty blastocysts were processed by the present method and 13 cell lines were established ( 16 %). subsequent to dissection of the inner cell mass , the inner cell mass cells are co - cultured with feeder cells to obtain a blastocyst - derived stem ( bs ) cell line . after obtaining the bs cell line , the cell line is optionally propagated to expand the amount of cells . thus , the present invention relates to a method as described above wherein the blastocyst - derived stem cell line is propagated . in one aspect , the invention relates to a method in which the propagation of blastocyst - derived stem cell line comprises passage of the stem cell line every 4 - 5 days . if the stem cell line is cultured longer than 4 - 5 days before passage , there is an increased probability that the cells undesirably will differentiate . a specific procedure of passaging the cells is given in example 5 herein . human bs cell lines may be isolated either from spontaneously hatched blastocysts or from expanded blastocysts with an intact zona pellucida . thus the present invention relates to a method as described above in which the blastocyst in step i ) is a spontaneously hatched blastocyst . for hatched blastocysts the trophectoderm may be left intact . either hatched blastocysts or blastocysts with a removed or partially removed zona pellucida may be put on inactivated feeder cells . zona pellucida of the blastocyst may be at least partially digested or chemically frilled prior to step ii ) e . g . by treatment with one or more acidic agents such as , e . g ., zd ™- 10 ( vitrolife , gothenburg , sweden ), one or more enzymes or mixture of enzymes such as pronase . a brief pronase ( sigma ) treatment of blastocysts with an intact zona pellucida results in the removal of the zona . other types of proteases with the same or similar protease activity as pronase may also be used . the blastocysts can be plated onto said inactivated feeder cells following the pronase treatment . in an embodiment of the invention step ii ) and / or step iv ) may be performed in an agent that improves the attachment of the blastocysts and / or if relevant the inner cell mass cells to the feeder cells . a suitable medium for plating the blastocysts onto feeder cells can be bs - medium that may be complemented with hyaluronic acid , which seems to promote the attachment of the blastocysts on the feeder cells and growth of the inner cell mass . hyaluronan ( ha ) is an important glycosaminoglycan constituent of the extracellular matrix in joints . it appears to exert its biological effects through binding interactions with at least two cell surface receptors : cd44 and receptor for ha - mediated motility ( rhamm ), and to proteins in the extracellular matrix . the positive effects of ha during the establishment of hbs cells may be exerted through its interactions with the surfactant polar heads of phospholipids in the cell membrane , to thereby stabilize the surfactant layer and thus lower the surface tension of the inner cell mass or blastocyst which may result in increased efficiency in binding to the feeder cells . alternatively , ha may bind to its receptors on the inner cell mass or blastocyst and / or to the feeder cells and exert biological effects which positively influence the attachment and growth of the inner cell mass . according to this , other agents that may alter the surface tension of fluids , or in other ways influence the interaction between the blastocyst and feeder cells can also be used in instead of hyaluronic acid . the inventors have also found that the culturing of the feeder cells is of importance for the establishment of the hbs cell line . in one embodiment , the propagation of blastocyst - derived stem cell line comprises passage of the feeder cells at the most 3 times , such as e . g . at the most 2 times . suitable feeder cells for use in a method of the invention are embryonic feeder cells . in a method according to the invention the feeder cells employed in steps ii ) and iv ) are the same or different and originate from animal source such as e . g . any mammal including human , mouse , rat , monkey , hamster , frog , rabbit etc . feeder cells from human or mouse species are preferred . another important criterion for obtaining an hbs cell line fulfilling the general requirements are the conditions under which the blastocysts are cultured . the blastocyst - derived stem cell line may accordingly by propagated by culturing the stem cells with feeder cells of a density of less than about 60 , 000 cells per cm 2 , such as e . g . less than about 55 , 000 cells per cm 2 , or less than about 50 , 000 cells per cm 2 . in a specific embodiment , the propagation of blastocyst - derived stem cell line comprises culturing the stem cells with feeder cells of a density of about 45 , 000 cells per cm 2 . these values apply in those cases where mouse feeder cells are used and it is contemplated that a suitable density can be found for other types of feeder cells as well . based on the findings of the present inventors , a person skilled in the art will be able to find such suitable densities . in a method according to the invention , the feeder cells may be mitotically inactivated in order to avoid unwanted growth of the feeder cells . the blastocyst - derived stem cell line obtained by the present invention maintains selfrenewal and pluripotency for a suitable period of time and , accordingly it is stable for a suitable period of time . in the present context the term “ stable ” is intended to denote proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells . the stem cell line obtained by the present invention fulfils the general requirements . thus , the cell line i ) exhibits proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells , and ii ) exhibits normal euploid chromosomal karyotype , and iii ) maintains potential to develop into derivatives of all types of germ layers both in vitro and in vivo , and iv ) exhibits at least two of the following molecular markers oct - 4 , alkaline phosphatase , the carbohydrate epitopes ssea - 3 , ssea4 , tra 1 - 60 , tra 1 - 81 , and the protein core of a keratin sulfate / chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody gctm - 2 , and v ) does not exhibit molecular marker ssea - 1 or other differentiation markers , and vi ) retains its pluripotency and forms teratomas in vivo when injected into immuno - compromised mice , and vii ) is capable of differentiating . the undifferentiated hbs cells according to the present invention is defined by the following criteria ; they were isolated from human pre - implantation fertilized oocytes , i . e . blastocysts , and exhibit a proliferation capacity in an undifferentiated state when grown on mitotically inactivated feeder cells ; they exhibit a normal chromosomal karyotype ; they express typical markers for undifferentiated hbs cells , e . g . oct4 , alkaline phosphatase , the carbohydrate epitopes ssea - 3 , ssea4 , tra 1 - 60 , tra 1 - 81 , and the protein core of a keratin sulfate / chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody gctm - 2 , and do not show any expression of the carbohydrate epitope ssea - 1 or other differentiation markers . furthermore , pluripotency tests in vitro and in vivo ( teratomas ) demonstrate differentiation into derivatives of all germ layers . according to the above , the invention is an essentially pure preparation of pluripotent human bs cells , which i ) exhibits proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells ; ii ) exhibits normal euploid chromosomal karyotype ; iii ) maintains potential to develop into derivatives of all types of germ layers both in vitro and in vivo ; iv ) exhibits at least two of the following molecular markers oct - 4 , alkaline phosphatase , the carbohydrate epitopes ssea - 3 , ssea4 , tra 1 - 60 , tra 1 - 81 , and the protein core of a keratin sulfate / chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody gctm - 2 v ) does not exhibit molecular marker ssea - 1 or other differentiation markers , and vi ) retains its pluripotency and forms teratomas in vivo when injected into immuno - compromised mice , and vii ) is capable of differentiating . procedures for the detection of cell markers can be found in gage , f . h ., science , 287 : 1433 - 1438 ( 2000 ). these procedures are well known for the skilled person and include methods such as rt - pcr or immunological assays where antibodies directed against the cell markers are used . in the following , methods for detection of cell markers , hybridisation methods , karyotyping , methods for measuring telomerase activity and teratoma formation are described . these methods can be used to investigate whether the hbs cells obtained according to the present invention fulfil the above - mentioned criteria . the human bdp stem cells maintained in culture are routinely monitored regarding their state of differentiation . cell surface markers used for monitoring the undifferentiated bs cells are ssea - 1 , ssea - 3 , ssea - 4 , tra - 1 - 60 , tra - 1 - 81 . human bdp stem cells are fixed in 4 % pfa and subsequently permeabilized using 0 . 5 % triton x - 100 . after washing and blocking with 10 % dry milk the cells are incubated with the primary antibody . after extensive washes the cell are incubated with the secondary antibody and the nuclei are visualized by dapi staining . the activity of alkaline phosphatase is determined using a commercial available kit following the instructions from the manufacturer ( sigma diagnostics ). the mrna levels for the transcription factor octet is measured using rt - pcr and gene specific primer sets ( 5 ′- cgtgmgctggagmggagaagctg , 5 ′- cmgggccgcagcttacacatgttc ) and gapdh as housekeeping gene ( 5 ′- accacagtccatgccatcac , 5 ′- tccaccaccctgttgctgta ). in one round of fish one ore more chromosomes are being selected with chromosome specific probes . this technique allows numerical genetic aberrations to be detected , if present . for this analysis cts uses a commercially available kit containing probes for chromosome 13 , 18 , 21 and the sex chromosomes ( x and y ) ( vysis . inc , downers grove , ill ., usa ). for each cell line at least 200 nuclei are being analyzed . the cells are resuspended in camoy &# 39 ; s fixative and dropped on positively charged glass slides . probe lsi 13 / 21 is mix with lsi hybridization buffer and added to the slide and covered with a cover slip . probe cep x / y / 18 is mixed with cep hybridization buffer and added in the same way to another slide . denaturing is performed at 70 ° c . for 5 min followed by hybridization at 37 ° c . in a moist chamber for 14 - 20 h . following a three step washing procedure the nuclei are stained with dapi ii and the slides analyzed in an invert microscope equipped with appropriate filters and software ( cytovision , applied imaging ). karyotyping allows all chromosomes to be studied in a direct way and is very informative , both numerical and larger structural aberrations can be detected . in order to detect mosaicism , at least 30 karyotypes are needed . however , this technique is both very time consuming and technically intricate . to improve the conditions for the assay the mitotic index can be raised by colcemid , a synthetic analog to colchicin and a microtubule - destabilizing agent causing the cell to arrest in metaphase , but still a large supply of cells are needed ( 6 × 10 6 cells / analysis ). the cells are incubated in the presence of 0 . 1 μg / ml colcemid for 1 - 2 h , and then washed with pbs and trypsinized . the cells are collected by centrifugation at 1500 rpm for 10 min . the cells are fixed using ethanol and glacial acetic acid and the chromosomes are visualized by using a modified wrights staining . comparative genomic hybridization ( cgh ) is complementary to karyotyping . cgh gives a higher resolution of the chromosomes and is technically less challenging . isolated dna is nicktranslated in a mixture of dna , a4 , texas red - dutp / fitc 12 - dutp , and dna polymerase i . an agarose gel electrophoresis is performed to control the size of resulting dna fragments ( 600 - 2000 bp ). test and reference dna is precipitated and resuspended in hybridization mixture containing formamide , dextrane sulfate and ssc . hybridization is performed on denatured glass slides with metaphases for 3 days at 37 ° c . in a moist chamber . after extensive washing one drop of antifade mounting mixture ( vectashield , 0 , 1 μg / ml dapi ii ) is added and the slides covered with cover slips . slides are subsequently evaluated under a microscope and using an image analysis system . since a high activity has been defined as a criterion for bs cells 6 the telomerase activity is measured in the bs cell lines . it is known that telomerase activity successively decrease when the cell reaches a more differentiated state . quantifying the activity must therefore be related to earlier passages and control samples , and can be used as a tool for detecting differentiation . the method , telomerase pcr elisa kit ( roche ) uses the internal activity of telomerase , amplifying the product by polymerase chain reaction ( pcr ) and detecting it with an enzyme linked immunosorbent assay ( elisa ). the assay is performed according to the manufacturer &# 39 ; s instructions . the results from this assay shows typically a high telomerase activity (& gt ; 1 ) for bs cells . the cell lines retain their pluripotency and forms teratomas in vivo when injected into immuno - compromised mice . in addition , in vitro these cells can form bs cell derived bodies . in both of these models , cells characteristic for all germ layers can be found . one method to analyze if a human bs cell line has remained pluripotent is to xenograft the cells to immunodeficient mice in order to obtain tumors , teratomas . various types of tissues found in the tumor should represent all three germlayers . reports have showed various tissues in tumors derived from xenografted immunodeficient mice , such as striated muscle , cartilage and bone ( mesoderm ) gut ( endoderm ), and neural rosettes ( ectoderm ). also , large portions of the tumors consist of disorganized tissue . severe combined immunodeficient ( scid )- mice , a strain that lack b - and t - lymphocytes are used for analysis of teratoma formation . human bs cells are surgically placed in either testis or under the kidney capsule . in testis or kidney , bs cells are transplanted in the range of 10 000 - 100 000 cells . ideally , 5 - 6 mice are used for each cell line at a time . preliminary results show that female mice are more post - operative stable than male mice and that xenografting into kidney is as effective in generating tumors as in testis . thus , a female scid - mouse teratoma model is preferable . tumors are usually palpable after approximate 1 month . the mice are sacrificed after 14 months and tumors are dissected and fixed for either paraffin - or freeze - sectioning . the tumor tissue is subsequently analyzed by immunohistochemical methods . specific markers for all three germlayers are used . the markers currently used are : human e - cadherin for distinction between mouse tissue and human tumour tissue , α - smooth muscle actin ( mesoderm ), α - fetoprotein ( endoderm ), and β - iii - tubulin ( ectoderm ). additionally , hematoxylin - eosin staining is performed for general morphology . the hbs cell line obtained by the method according to the method of the present invention can be used for the preparation of differentiated cells . therefore the invention also relates to such differentiated cells . in a further embodiment , the hbs cell line according to the invention has the ability of differentiating into an insulin producing cells . they may be capable of forming islet - like structures , and the amount of insulin producing β - cells is generally higher than 25 %, such as e . g . higher than 35 %, or higher than 40 %, or higher than 45 %, or higher than 50 %. thus in one embodiment , the insulin producing cells produces at least about 300 ng insulin / mg total protein such as at least about 380 ng insulin / mg total protein or at least about 450 ng insulin / mg total protein . the blastocyst - derived stem cells may have the ability to differentiate into differentiated cells , which display the expression of pancreatic cell type markers , including at least one of insulin , glut - 2 , pdx - 1 , glucokinase , glucagon and somatostatin . alternatively the hbs cells have the ability to differentiate into insulin - producing cells characterized by their organization into islet - like structures comprising an inner core of β - cells surrounded by an outer layer of neuron - type cells , which neuron - type cells display expression of at least one of the following neuronal cell type markers , including neuron - specific β - iii tubulin ( tuj1 ), neun , doublecortin , tyrosine hydroxylase and map 2 . an object of the invention is also to provide an essentially pure preparation of bs stem cells that can be made to differentiate into oligodendrocytes , and also to provide an essentially pure preparation of oligodendrocytes prepared by this method . oligodendrocytes can be characterized by the presence of cell markers such s rip , galc or o4 . the blastocyst - derived stem cells that are capable of being made into differentiated cells may display the expression of at least one of the following neuronal cell type markers , including neuron - specific β - iii tubulin ( tuj1 ), neun , doublecortin , tyrosine hydroxylase and map 2 . in a still further aspect , the invention relates to the use of a preparation of differentiated cells derived from the blastocyst - derived stem cells obtained by a method according to the invention for the manufacture of a medicament for the prevention or treatment of pathologies or diseases caused by tissue degeneration . a further object of the invention is to provide cells that may be used for the preparation of a medicament for treating and / or preventing diseases that may be cured by “ cell genesis ”. by the term “ cell genesis ” is meant the generation of new cells such as neurons , oligodendrocytes , schwann cells , astroglial cells , all blood cells , chondrocytes , cardiomyocytes , oligodendroglia , astroglia , and / or different types of epithelium , endothelium , liver -, kidney -, bone -, connective tissue -, lung tissue -, exocrine and endocrine gland tissue - cells . in an embodiment , the invention relates to the use of a preparation of differentiated cells derived from the blastocyst - derived stem cells obtained for the manufacture of a medicament for the prevention or treatment of pathologies or diseases in the pancreas such as diabetes including diabetes type i . the differentiated cells derived from the blastocyst - derived stem cell line obtained may also be used for the manufacture of a medicament for the prevention or treatment of pathologies or diseases in the nervous system . such diseases include multiple schlerosis , spinal chord injury , encephalopathies , parkinson &# 39 ; s disease , huntingdon &# 39 ; s disease , stroke , traumatic brain injuries , hypoxia induced brain injuries , ischemia induced brain injuries , hypoglycemic brain injuries , degenerative disorders of the nervous system , brain tumors and neuropathies in the peripheral nervous system . in a still further embodiment , the invention relates to a kit for performing the method according to the invention . the kit comprises at least a first and a second component in separate compartments . the components comprise an agent that improves the attachment of the blastocysts , a digestive agent , bs - cell medium and / or feeder cells or mixtures thereof . the kit may further comprise blastocysts with an intact zona pelludica or spontaneously hatched blastocysts . in another aspect , the invention relates to a method for producing an essentially pure preparation of insulin - producing differentiated stem cells , comprising the steps of ; i ) expanding human blastocyst - derived stem cells by growing these on an inactivated feeder cell layer in a suitable medium ; ii ) generating blastocyst - derived stem cell bodies by dissociating colonies formed in step i ) into smaller aggregates or individual cells , followed by transferring said aggregates or individual cells to non - adherent containers where they are incubated in a suitable medium ; iii ) plating the blastocyst - derived stem cell bodies in containers in a suitable medium ; iv ) selecting nestin - positive neural precursors in itfsn medium ; v ) expanding pancreatic endocrine progenitor cells in , n2 - medium comprising b27 media complement and basic fibroblast growth factor ; vi ) changing the medium to a basic fibroblast growth factor - free n2 medium . the manual dissection may be performed by using glass capillaries as a cutting tool . the human blastocyst - derived stem cells employed in the above - mentioned method are typically those obtained as described herein . more specifically the medium used in step i ) is human blastocyst - derived stem cell medium , the medium used in step ii ) is blastocyst - derived stem cell body medium , and the medium used in step iii ) is blastocyst - derived stem cell body medium . a kit according to the invention may also be applied to the above - mentioned method . in this case , the kit comprises at least two of the following components in separate compartments ; mitomycin c , hbs medium , bs cell body medium , itsfn - medium , n2 - medium , b27 - media supplement , nicotinamide , and bfgf . the kit may further comprise an essentially pure human blastocyst - derived stem cell line obtained by the method according to the present invention . fig1 : blastocyst ( before pronase treatment ) from which human bs cell line 167 was established . fig2 : blastocyst ( after pronase treatment ) from which human bs cell line 167 was established . fig3 : blastocyst 167 two days after plating on embryonic mouse fibroblasts . fig4 : human bs cells at passage 69 cultured on embryonic mouse fibroblasts . fig5 : human bs cells at passage 71 cultured on embryonic mouse fibroblasts . fig8 : expression of molecular markers for undifferentiated human bs cells . ( a ) rt - pcr analysis of total rna extracted from undifferentiated ( ud ) and from differentiated ( d ) human bs cells for the presence of oct - 4 , insulin , glut - 2 , glucagon , and pdx - 1 mrna . in controls the reverse transcriptase was omitted (- rt ). β - actin serves as housekeeping gene . ( b ) shows the presence of alkaline phosphatase by immunostaining in undifferentiated human bs cell colonies . ( c ) analysis of ssea - 1 expression by immunostaining of undifferentiated human bs cell colonies . ( d ) undifferentiated bs cells were immunopositive for ssea - 3 ( data not shown ) and ssea - 4 . ( e ) immunopositive human bs cell colonies for tra - 1 - 60 and in ( f ) for tra - 1 - 81 showing their undifferentiated status . magnification 40 ×. fig1 . human bs cells differentiate in vitro into all germ layer cell types . corresponding fluorescent micrographs show immunopositive cells stained with germ layer specific markers after 10 days in vitro . ( a and b ) show examples of neuroectodermal cells expressing nestin for neuronal precursors ( a ) and β - iii - tubulin for postmitotic neurons ( b ) while ( c ) shows examples of mesodermal cells immunoreactive for desmin ; ( d ) examples of cells expressing α - fetoprotein . fig1 . immuno staining for nestin in in vitro differentiated human bs cells . fig1 . immuno staining for insulin in in vitro differentiated human bs cells . fig2 . immuno staining for β - iii - tubulin in in vitro differentiated human bs cells . as used herein , the term “ blastocyst - derived stem cell ” is denoted bs cell , and the human form is termed “ hbs cells ”. as used herein , the term “ blastocyst - derived stem cell bodies ” is denoted “ bs cell bodies ”. as used herein , the term “ ef cells ” means “ embryonic fibroblast feeder ”. these cells could be derived from any mammal , such as mouse or human . one suitable medium used in the invention is termed “ bs - cell medium ” or “ bs - medium ” and may be comprised of ; knockout ® dulbecco &# 39 ; s modified eagle &# 39 ; s medium , supplemented with 20 % knockout ® serum replacement and the following constituents at their respective final concentrations : 50 units / ml penicillin , 50 μg / ml streptomycin , 0 , 1 mm non - essential amino acids , 2 mm l - glutamine , 100 μm β - mercaptoethanol , 4 ng / ml human recombinant bfgf ( basic fibroblast growth factor ). another suitable medium for the present invention is “ bs cell body medium ”, this may be comprised as follows ; knockout ® dulbecco &# 39 ; s modified eagle &# 39 ; s medium , supplemented with 20 % knockout ® serum replacement and the following constituents at their respective final concentrations : 50 units / ml penicillin , 50 pug / ml streptomycin , 0 , 1 mm nonessential amino acids , 2 mm l - glutamine and 100 μm β - mercaptoethanol ( itskovitz - eldor , j . et al ., 2000 ). in the present context the term “ stable ” is intended to denote proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells . the invention will now be described with reference to the following examples . the examples are included herein for illustrative purposes only and are not intended to limit the scope of the invention in any way . the general methods described herein are well known to a person skilled in the art and all reagents and buffers are readily available , either commercially or easily prepared according to well - established protocols in the hands of a person skilled in the art . all incubations were in 37 ° c ., under a co 2 atmosphere . establishment of an essentially pure preparation of undifferentiated stem cells from spontaneously hatched blastocysts human blastocysts were derived from frozen or fresh human in vitro fertilized embryos . spontaneously hatched blastocysts were put directly on feeder cells ( ef ) in bs cell medium ( knockout dulbecco &# 39 ; s modified eagle &# 39 ; s medium , supplemented with 20 % knockout serum replacement , and the following constituents at the final concentrations : 50 units / ml penicillin , 50 μg / ml streptomycin , 0 . 1 mm non - essential amino acids , 2 mm l - glutamine , 100 μm β - mercaptoethanol , 4 ng / ml human recombinant bfgf ( basic fibroblast growth factor ), supplemented with 0 . 125 mg / ml hyaluronic acid . after plating the blastocysts on the ef cells , growth was monitored and when the colony was large enough for manual passaging approximately 1 - 2 weeks after plating ) the inner cell mass cells were dissected from other cell types and expanded by growth on new ef cells . establishment of an essentially pure preparation of undifferentiated stem cells from blastocysts with an intact zona pellucida for blastocysts with an intact zona pellucida , a brief pronase ( 10 u / ml , sigma ) incubation in rs2 ( icm - 2 ) medium ( vitrolife , gothenburg , sweden ) was used to digest the zona , after which the blastocyst was put directly on the ef cell layer in bs medium supplemented with hyaluronic acid ( 0 . 125 mg / ml ). the cells were harvested for rt - pcr and histological ( alkaline phosphatase ) and immunocytochemical analysis ( see below ). rna isolation and rt - pcr . total cellular rna was prepared using rneasy mini kit ( qiagen ) according to the manufacturer &# 39 ; s recommendations . the cdna synthesis was carried out using amv first strand cdna synthesis kit for rt - pcr ( roche ) and pcr using platinum taq dna polymerase ( invitrogen ). histochemical staining for alkaline phosphatase was carried out using commercially available kit ( sigma ) following the manufacturer &# 39 ; s recommendations . mouse embryonic fibroblasts feeder cells were cultivated on tissue culture dishes in emfi - medium : dmem ( dulbecco &# 39 ; s modified eagle &# 39 ; s medium ), supplemented with 10 % fcs ( fetal calf serum ), 0 , 1 μm β - mercaptoehanol , 50 units / ml penicillin , 50 μg / ml streptomycin and 2 mm l - glutamine ( gibcobrl ). the feeder cells were mitotically inactivated with mitomycin c ( 10 μg / ml , 3 hrs ). human bs cell - colonies were expanded by manual dissection onto inactivated mouse embryonic fibroblasts feeder cells . human bs cells were cultured on mitotically inactivated mouse embryonic fibroblasts feeder cells in tissue culture dishes with bs - cell medium : knockout ® dulbecco &# 39 ; s modified eagle &# 39 ; s medium , supplemented with 20 % knockout ® serum replacement and the following constituents at their respective final concentrations : 50 units / ml penicillin , 50 μg / ml streptomycin , 0 , 1 mm non - essential amino acids , 2 mm l - glutamine , 100 μm β - mercaptoethanol , 4 ng / ml human recombinant bfgf ( basic fibroblast growth factor ). seven days after passage the colonies were large enough to generate bs cell bodies . bs cell colonies were cut with glass capillaries into 0 . 4 × 0 . 4 mm pieces and plated on non - adherent bacterial culture dishes containing bs cell body medium : knockout ® dulbecco &# 39 ; s modified eagle &# 39 ; s medium , supplemented with 20 % knockout ® serum replacement and the following constituents at their respective final concentrations : 50 units / ml penicillin , 50 μg / ml streptomycin , 0 , 1 mm non - essential amino acids , 2 mm l - glutamine and 100 μm β - mercaptoethanol ( itskovitz - eldor , j . et al ., 2000 ). the bs cell bodies , including cystic bs cell bodies , formed over a 7 - 9 - day period . before passage the hbs cells are photographed using a nikon eclipse te2000 - u inverted microscope ( 10 × objective ) and a dxm 1200 digital camera . colonies are passaged every 4 - 5 days . the colonies are big enough to be passaged when they can be cut in pieces ( 0 . 1 - 0 . 3 × 0 . 1 - 0 . 3 mm ). the first time the cells are passaged , they have grown for 1 - 2 weeks and can be cut in approximately four pieces . the colonies are focused , one by one , in a stereo - microscope and cut in a checkered pattern according to the size above . only the inner homogeneous structure is passaged . each square of the colony is removed with the knife , aspirated into a capillary and placed on new feeder cells ( with the maximum age of 4 days ). 10 - 16 squares are placed evenly in every new ivf - dish . the dishes are left five to ten minutes so the cells can adhere to the new feeder and then placed in an incubator . the hbs medium is changed three times a week . if the colonies are passaged , medium is changed twice that particular week . normally a “ half change ” is made , which means that only half the medium is aspirated and replaced with the equal amount of fresh , tempered medium . if necessary the entire volume of medium can be changed . colonies with the appropriate undifferentiated morphology from the cell line are cut as for passage . 100 - 200 ml liquid nitrogen is sterile filtered into a sufficient amount of cryotubes . two solutions a and b are prepared ( a : 800 μl cryo pbs with 1 m trehalose , 100 μl etylen glycole and 100 μl dmso , b : 600 μl cryo pbs with 1 m trehalose , 200 μl etylen glycole and 200 μl dmso ) and the colonies are placed in a for 1 minute and in b for 25 seconds . closed straws are used to store the frozen colonies . after the colonies have been transferred to a straw , it is immediately placed in a cryotube with sterile filtered nitrogen . the cells are inactivated with emfi medium containing mitomycin c by incubation at 37 ° c . for 3 hours . ivf - dishes are coated with gelatin . the medium is aspirated and the cells washed with pbs . pbs is replaced with trypsin to detach the cells . after incubation , the trypsin activity is stopped with emfi medium . the cells are then collected by centrifugation , diluted 1 : 5 in emfi medium , and counted in a bürker chamber . the cells are diluted to a final concentration of 170k cells / ml emfi medium . the gelatin in the ivf - dishes is replaced with 1 ml cell suspension and placed in an incubator . emfi medium is changed the day after the seeding . 1 . itskovitz - eldor , j . et al . differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers . mol med 6 , 88 - 95 . 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( 2001 ). 6 . brewer , g . j ., torricelli , j . r ., evege , e . k . & amp ; price , p . j . optimized survival of hippocampal neurons in b27 - supplemented neurobasal , a new serum - free medium combination . j neurosci res 35 , 567 - 76 . ( 1993 ). 7 . otonkoski , t ., beattie , g . m ., mally , m . i ., ricordi , c . & amp ; hayek , a . nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells . j clin invest 92 , 1459 - 66 . ( 1993 ). 8 . assady , s ., maor , g ., michal , a ., itskovitz - eldkor , j ., skkorecki , k . l . & amp ; tzukerman , m ., insulin production by human embryonic stem cells . diabetes 50 , 1691 - 1697 , 2001 . 9 . gardner et al , embryo culture systems , in trounson , a o ., and gardner , d . k . ( eds ), handbook of in vitro fertilization , second edition . crc press , boca raton , pp . 205 - 264 ; 10 . gardner et al , fertil steril , 74 , suppl 3 , o - 086 ; 12 . gardner et al , j reprod immunol , in press ; 13 . hooper et al , biol reprod , 62 , suppl 1 , 249 .