WO2007082963A1 - Human embryo stem-cell lines and methods for using same - Google Patents

Human embryo stem-cell lines and methods for using same Download PDF

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WO2007082963A1
WO2007082963A1 PCT/ES2006/000017 ES2006000017W WO2007082963A1 WO 2007082963 A1 WO2007082963 A1 WO 2007082963A1 ES 2006000017 W ES2006000017 W ES 2006000017W WO 2007082963 A1 WO2007082963 A1 WO 2007082963A1
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population
cells
embryonic stem
human
human embryonic
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PCT/ES2006/000017
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Spanish (es)
French (fr)
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Carlos SIMÓN VALLÉS
Antonio Pellicer Martinez
Rubén MORENO PALANQUES
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Fundación Instituto Valenciano De Infertilidad
Centro De Investigación Príncipe Felipe
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Priority to PCT/ES2006/000017 priority Critical patent/WO2007082963A1/en
Priority to US11/655,048 priority patent/US20070202595A1/en
Publication of WO2007082963A1 publication Critical patent/WO2007082963A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)

Definitions

  • the invention relates to human embryonic cell lines and methods for using them.
  • CMEh Human embryonic stem cells
  • melanocytes hematopoietic cells
  • hepatocytes hepatocytes
  • renal cells hepatocytes
  • skeletal muscle cells dopaminergic neurons
  • Ia glia hepatocytes
  • cardiomyocytes endothelial cells
  • osteoblasts a cell lineages in vitro and in vivo
  • CMEh cells are therapeutically attractive due to their pluripotence. The use of such cells and their differentiated progeny are contemplated for the treatment of various conditions.
  • CMEh transplantation of CMEh (and its differentiated progeny) in human subjects will require, in the majority of cases, histocompatibility between the CMEh and the human receptors of such cells. Given the level of diversity in human histocompatibility antigens, it has been calculated that 150,000 lines would need to be generated.
  • the invention starts from the premise, in part, of the derivation and characterization of CMEh lines from long-term cryopreserved human embryos.
  • the lines were derived in a defined medium without serum and without animal feeder cells (i.e., animal-free conditions). Two examples of these lines are designated VAL-1 and VAL-2. These lines were derived from "brother" embryos (ie, embryos that have identical origins) and are immunologically identical. Consequently, these lines can be used together, for example, in a therapeutic practice, due to their histocompatibility.
  • the invention provides an isolated population of human embryonic stem cells in which said population of cells (1) has a normal karyotype after at least 85 passes; (2) expresses the phase-specific embryonic antigen (SSEA) 4, the tumor rejection antigen (TRA) -I-60 and TRA-1-81; (3) is positive for alkaline phosphatase; (4) expresses Oct-4, Rex-1, Crypto, Thy-1 and Nanog; (5) is negative for Matni, Amylase and Dbh; and (6) has telomerase activity.
  • SSEA phase-specific embryonic antigen
  • TRA tumor rejection antigen
  • the invention provides an isolated population of human embryonic stem cells, in which said population of cells has the characteristics of the VAL-1 or VAL-2 embryonic stem cell line.
  • the invention provides an isolated population of embryonic stem cells, in which said population of cells is the VAL-1 or VAL-2 embryonic stem cell line, and the progeny of the same.
  • the progeny can be differentiated or undifferentiated progeny including, but not limited to, cardiac, neuronal, muscular and hematopoietic progeny.
  • the population comprises genetically modified cells.
  • the population is subjected to passes at least 50 times, at least 75 times, at least 100 times, or more. In one embodiment, the population is subjected to passes at least 85 times or at least 120 times.
  • the population has been cryopreserved and optionally thawed and subjected to passes, without changes in the expression of the marker, the karyotype or the telomerase activity.
  • the population can be cryopreserved for more than 1 year.
  • the "populations" of human embryonic stem cells mentioned herein are also called “lines” of human embryonic stem cells.
  • the invention provides a pair of populations (or lines) isolated from human embryonic stem cells that express the markers of HLA (human leukocyte antigen) A2, A23, B44, B40, CW4, CW5, DR7, DR15, DQ2 and DQ6.
  • the invention provides compositions and methods for using the pair of human embryonic stem cell populations (and / or their differentiated progeny).
  • the pair of lines (and / or their differentiated progeny) are used in human subjects who have one or more of the HLA markers mentioned above.
  • the populations (and / or their differentiated progeny) can be introduced into the subjects at the same time or at different times.
  • the methods may further comprise the use of different populations differentiated from the lines in the same subject.
  • a subject can receive hematopoietic cells derived from VAL-1 and keratinocytes derived from VAL-2 at the same time or at different times.
  • the invention provides compositions comprising one or more human embryonic stem cells of any of the previous populations of human embryonic stem cells.
  • the composition may be a pharmaceutical preparation, but it is not limited thereto. - TO -
  • the invention provides a culture comprising any of the previous populations of stem cells.
  • the culture may comprise feeder cells, preferably human feeder cells such as, but not limited to, human placental feeder cells.
  • feeder cells are mitotically inactivated such as, for example, by irradiation.
  • the culture may be a serum free culture.
  • the culture may comprise fibroblast growth factor (FGF) such as, but not limited to the basic FGF (FGFb).
  • FGF fibroblast growth factor
  • the culture may be free of feeder cells.
  • the culture is free of animal products (ie, non-human).
  • the culture may comprise human embryonic stem cells and the differentiated progeny thereof.
  • the invention provides a method for cultivation that includes the propagation of human embryonic stem cell populations comprising culturing any of the previous populations of human embryonic stem cells in a serum-free medium and optionally in the presence of human feeder cells.
  • Human feeder cells can be human placental feeder cells. Feeder cells can be inactivated mitotically, for example, by irradiation.
  • the medium may comprise FGF such as, but not limited to, FGFb. In some embodiments, the FGFb is present in an amount of about 1-15 ng / mL or 1-10 ng / mL.
  • the invention provides a method for differentiating in vitro a population of human embryonic stem cells comprising exposing any of the previous populations of human embryonic stem cells to differentiation conditions for a time sufficient to allow the differentiation of the population of human embryonic stem cells in differentiated cells.
  • the differentiation conditions may comprise feeder cells or may be free from feeder cells.
  • the differentiation conditions comprise one or more factors selected from the group consisting of retinoic acid, a factor of epidermal growth (EGF), bone morphogenetic protein 4 (BMP4), fibroblast growth factor (FGF), steroid hormones, activin-A, transforming growth factor beta 1 (TGF- / J1), hepatocyte growth factor (HGF ) and nerve growth factor (NGF).
  • the method may further comprise introducing the differentiated cells into a subject such as, but not limited to, a human subject.
  • the invention provides a method for differentiating a population of human embryonic stem cells that comprises introducing any of the previous populations of human embryonic stem cells into a subject including, but not limited to, a human subject.
  • the subject has a state that affects the liver, muscle, skin, brain, nervous system, heart, circulatory system, hematopoietic system, pancreas or bone.
  • the population is introduced into the subject by local administration, such as, but not limited to, administration to an organ or tissue.
  • the population is introduced into the subject by systemic administration, such as, but not limited to, intravenous administration.
  • the method further comprises exposing the population to differentiation conditions in vitro before the introduction into the subject.
  • the subject expresses one, two, three, four, five, six, seven, eight or nine HLA markers selected from the group consisting of A2, A23, B44, B40, CW4, CW5, DR7, CD15, DQ2 and DQ6. In some embodiments, the subject expresses all the previous markers. In some embodiments, the subject receives the VAL-1 and VAL-2 cells or the progeny thereof.
  • the invention provides a method for determining the therapeutic efficacy or cytotoxicity of a compound comprising exposing any of the previous lines of human embryonic stem cells to a compound (for example, by contacting the line with the compound) , and determine an effect of the compound on the human embryonic stem cell line.
  • the line can be differentiated before contacting the compound.
  • the differentiation can be hematopoietic differentiation or neuronal differentiation, but the invention is limited to them.
  • the effect of the compound can be cell death, cell growth (for example, increase in the number of cells), the increase in the number of undifferentiated human embryonic stem cells, the decrease in the number of undifferentiated human embryonic stem cells (for example, due to differentiation), the increase in the number of cells of a particular differentiated lineage, changes in expression profiles, and the like.
  • Figures 1A-F are a series of photographs that show the morphological characteristics of the derivation of VAL-1 and VAL-2.
  • Two different embryos (A, D) were thawed and adhered to human placental fibroblasts. After 18 (B) and 21 (E) days of shunt, the cell colonies had the typical appearance of the CMEh, which was maintained after at least 90 days of cell culture (C, F).
  • Figures 2A-J show several characteristics of VAL-1 and VAL-2, including karyotypes (A, F), immunostaining for determining the specific embryonic antigen of phase 4 (B, G), TRA-1-60 (C , H) and TRA-1-81 (D, I), and the alkaline phosphatase assay (E, J). Both cell lines had normal karyotypes and were positive for all tested markers of non-differentiation (i.e., immature).
  • Figure 3 shows the telomerase activity of the lines of CMEh VAL-
  • the negative control was obtained by thermal inactivation in both lines.
  • the 36 bp band corresponds to the internal control of the polymerase chain reaction and the 50 bp band corresponds to the telomerase activity, which increases in 6 bp bands in the immortal cells.
  • Figure 4 is a list of GeneScan MR showing the genetic fingerprint profiles of donor samples from VAL-1 (third row), VAL-2 (fourth row), maternal (egg donor) (first row) and paternal ( sperm donor) (second row).
  • Figure 5 is a list of GeneScan MR showing the genetic fingerprint profiles of VAL-1 and VAL-2. The figure shows a detailed analysis of loci D351358 and VWA of the third and fourth rows of Figure 4.
  • Figure 6 is a genomic profile comparison of VAL-1 and VAL-2 that shows that the lines are not genetically identical to each other. Data points surrounded by circles represent varying or different genomic loci levels between the lines.
  • Figures 7A-D are photographs of differentiated cardiomyocytes spontaneously derived from VAL-1.
  • Figures 8A-C are photographs of spontaneously differentiated immunotened progeny of VAL-1 and VAL-2.
  • Figure 8B shows the expression of tubulin B in neuron-like cells.
  • Figure 8C shows the expression of actin in cells similar to those of muscle.
  • Figures 9A-D are photographs of teratomas formed after the intrathestic injection of VAL-1 and VAL-2 in SCID mice.
  • FIGS. 10A-L are photographs showing the histology of a teratoma derived from VAL-1. It should be understood that the drawings are not necessary to enable the invention.
  • the invention starts from the premise, in part, of the derivation, characterization and use of CME lines from human embryos. These lines are derived from human embryos that have been cryopreserved for several years.
  • the invention provides a molecular and functional characterization of these lines and thereby encompasses embryonic stem cell lines that have similar characteristics.
  • the invention also encompasses methods of handling such lines, including cultivation (which includes propagation), long-term storage (which includes cryopreservation), tests (for example, in a detection test), and the differentiation of such lines.
  • the CMEh lines were generated from embryos with identical origin (i.e., obtained from the same progenitors (i.e., sources of ovules and sperm)). Consequently, embryos and lines are referred to herein as "brothers" lines and embryos. In addition, however, the lines are also immunologically identical to each other, as described herein. As a result, the invention provides for the combined use of such lines in treatment regimens given their histocompatibility.
  • the invention provides CME lines of human origin.
  • a CME line is a cell line derived in vitro from an embryo and having properties similar to those of stem cells.
  • the properties of the stem cells of the CMEh lines of the invention include one or more of the genotypic, phenotypic and functional properties described herein.
  • the line maintains such properties in the long term and through several passes, cultures and freezing and thawing cycles.
  • VAL-1 has undergone passes at least 120 times
  • VAL-2 has undergone passes at least 85 times while maintaining its phenotypic and functional properties.
  • the properties Genotypic can be global, such as the karyotype, or more detailed, such as genomic profiles.
  • the lines provided herein preferably contain a normal karyotype.
  • Phenotypic properties include morphological properties such as a small round cell shape with a high nucleus ratio with respect to cytoplasm and prominent nucleoli. Phenotypic properties also include the expression of intracellular markers or cell surface. These properties include the expression of immature or non-differentiation markers, such as the phase 4 specific embryonic antigen (SSEA 4), the keratin sulfate associated antigens, the tumor rejection antigen (TRA) -I-60 and TRA-1 -81, Oct-4, Rex-1, Crypto, Thy-1 and / or Nanog cell transcription factor.
  • SSEA 4 phase 4 specific embryonic antigen
  • TRA tumor rejection antigen
  • the CMEh lines can also be characterized according to the non-expression of certain markers such as differentiation markers.
  • the lines provided in this document are characterized as not expressing the differentiation markers Matni, Amylase and Dbh.
  • Other phenotypic properties include the presence of alkaline phosphatase and telomerase.
  • Phenotypic markers can be tested in several ways. For example, they can be tested using specific marker binding agents, such as specific marker antibodies or fragments thereof.
  • SSEA-4 can be detected by immunostaining using monoclonal antibodies such as MC-813-70 (Salter and Knowles, 1979) that is commercially available from Chemicon (Temecula, CA).
  • TRA-1-60 and TRA-1-81 expression can be detected using monoclonal antibodies also available from commercial sources such as Chemicon (Temecula, CA). Nanog can also be detected using antibodies such as AF1997 available from R&D Systems (Minneapolis, MN). Oct-4 can also be detected using antibodies such as AF1759 from R&D Systems (Minneapolis, MN), or sc-8628 or sc-9081 available from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phenotypic markers can also be tested using reverse transcriptase - polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase - polymerase chain reaction
  • Markers such as Oct-4, Rex-1, Crypt, Thy-1, Nanog, Matni, Amylase and Dbh can be detected using RT-PCR.
  • the primers for use in the amplification by means of the RT-PCR of these various markers are known in the art. (See, for example, Brimble et al. Stem CeIIs and Development, 13: 585-596, 2004; Noaksson et al. Stem CeIIs, 23: 1460-1467, 2005.)
  • such primers can be designed by a regular expert in The technique using routine experimentation.
  • the primers can be designed using software based on the network available for free (Primer3, Genefisher).
  • markers can still be detected using enzymatic assays.
  • markers include alkaline phosphatase and telomerase.
  • Alkaline phosphatase can be detected in a conventional assay using reagents available from commercial sources such as Chemicon (Temecula, CA) or Vector Laboratories (Burlingame, CA; Vector Blue / Red Alkaline Phosphatase Substrate Kit).
  • Telomerase activity can be detected using commercially available telomerase detection kits, such as those available from Chemicon (TRAPEZE Telomerase Detection Kit, Temecula, CA), optionally together with DNA gel staining, such as, but not limited to, staining in SYBR gel (Molecular Probes, Eugene, OR).
  • TRAPEZE Telomerase Detection Kit Temecula, CA
  • DNA gel staining such as, but not limited to, staining in SYBR gel (Molecular Probes, Eugene, OR).
  • the functional properties of the CMEh include the ability to form compact colonies in vitro, the ability to grow in the long term in vitro and, more importantly, the ability to maintain the characteristics of the stem cells in the long term, such as, but not limited to, pluripotence, as described herein. These properties can be tested visually, or by culture and / or differentiation in vitro or in vivo.
  • the CMEh are at least pluripotent and, in some cases, they can be totipotent. Pluripotence refers to the ability of these cells to generate most, if not all, of the tissues in an organism. Totipotence refers to the ability of these cells to generate a whole organism
  • the CMEh of the invention can be differentiated into mesoderm, endoderm and ectoderm lineages.
  • CMEh can differentiate into at least one mesoderm lineage such as bone, cartilage, smooth muscle, heart muscle, skeletal muscle, kidney, striated muscle and hematopoietic cells; at least one endoderm lineage such as liver, pancreas, thyroid gland, primitive intestine and respiratory epithelium; and at least one ectoderm lineage such as skin, pigment cells such as melanocytes, neurons, glia cells, hair follicles and dental buds. CMEh can also differentiate into germ cells.
  • mesoderm lineage such as bone, cartilage, smooth muscle, heart muscle, skeletal muscle, kidney, striated muscle and hematopoietic cells
  • endoderm lineage such as liver, pancreas, thyroid gland, primitive intestine and respiratory epithelium
  • ectoderm lineage such as skin, pigment cells such as melanocytes, neurons, glia cells, hair follicles and dental buds.
  • CMEh can also differentiate into germ cells.
  • the CMEh lines may exist in culture for prolonged periods of time (for example, up to a year or more, and potentially indefinitely) without fully differentiating and without depletion, and maintaining their original phenotype, for example as described in the present document Consequently, the invention provides CMEh lines that have one or more of the above characteristics.
  • the CMEh lines Preferably, the CMEh lines have all the above characteristics, however, the invention is not limited to them.
  • the CMEh lines provided by the invention include those designated VAL-1 and VAL-2.
  • VAL-1 and VAL-2 are sister cell lines in the sense that they are derived from sister embryos (that is, embryos that have the same "parents"). Marker genetic fingerprint profiles for parents and cell lines are shown in Figure 4.
  • Three different polymorphic markers that have independent hereditary transmission were analyzed (ie, D3S1358, vWA, FGA, D8D1179, D21S11, D18S51, D5S818, D13S317, D7S820).
  • the data demonstrate, with a 99% probability, that the VAL-1 and VAL-2 cell lines were derived from the source of maternal and paternal samples shown, and consequently that VAL-1 and VAL-2 are sister cell lines.
  • VAL-1 and VAL-2 are also immunologically identical.
  • the lines share identical HLA markers A2, A23, B40, B44, CW4, CW5, DR7, DR15, DQ2 and DQ6.
  • the lines are genetically different, such as demonstrated by the comparison of genomic profile of Figure 6. Therefore, cells can be used to determine the importance of such genetic differences on the functionality of the lines, such as, but not limited to, the ability to differentiate.
  • the invention provides the CMEh lines in an isolated form.
  • isolated means that the cells are physically separated from their natural environment, such as a blastocyst, an MCI (internal cell mass) and components thereof.
  • the lines are generally provided as a clonal population.
  • the CMEh lines of the invention can be further characterized by the method of their derivation.
  • VAL-1 and VAL-2 were derived from long-term cryopreserved embryos (that is, 5 years or more) in the blastocyst phase. Human embryos on day 2, cryopreserved and donated for research, were frozen and then treated to remove the zona pellucida using, for example, Tyrode acid solution.
  • the embryos were washed in medium (for example, 80% DMEM, 20% serum-free deficient serum replacement (GibcoBRL), optionally supplemented with 0.1 mM non-essential amino acids, 0.1 mM beta-mercaptoethanol and L- 1 mM glutamine), which optionally contained 12 ng / mL basic human fibroblast growth factor (Invitrogen).
  • medium for example, 80% DMEM, 20% serum-free deficient serum replacement (GibcoBRL), optionally supplemented with 0.1 mM non-essential amino acids, 0.1 mM beta-mercaptoethanol and L- 1 mM glutamine), which optionally contained 12 ng / mL basic human fibroblast growth factor (Invitrogen).
  • the embryos were then transferred onto human feeder cells.
  • Suitable human feeder cells include, but are not limited to, human placental fibroblasts. These feeder cells were inactivated mitotically. Mitotic activation can be carried out using, for example, i
  • the cultures were maintained for 2-3 weeks, at which time the initial colonies were mechanically altered and allowed to rejoin the feeder cells. After about a week, the colonies were mechanically altered again and then transferred to fresh feeder cells. CMEhs were morphologically identified as round cells with prominent nucleoli. The individual colonies were isolated and re-cultivated in order to achieve a clonal population, after Io that the lines could be submitted to at least 50, 75, 100, or more passes.
  • the invention contemplates the derivation of the CMEh lines provided herein in other ways.
  • the CMEh lines can be generated from newly prepared embryos or from embryos that have been cryopreserved for only a short time (for example, days, weeks or months).
  • the derivation process can involve the isolation of the MCI cells from a blastocyst without treatment with Tyrode acid solution.
  • compositions comprising the CMEhs provided herein.
  • Such compositions may comprise other components such as human feeder cells (eg, human placental feeder cells), progeny of stem cell lines, including differentiated progeny, differentiation factors, extracellular matrices, pharmaceutically acceptable carriers and the like.
  • These compositions may include cultures of the CMEh.
  • Such cultures may include human or animal serum, or they may be serum free.
  • cultures may comprise serum replacements, as described herein.
  • the invention also contemplates cultivation methods that include the propagation of undifferentiated CMEh lines, optionally for weeks, months or years. As discussed in the examples, these culture methods and conditions are similar to the derivation methods and conditions provided herein.
  • the lines can be cultured in the presence of feeder cells, preferably human feeder cells, and even more preferably human placental feeder cells, as described by Genbacev et al. Fertile Steril. 2005, 83: 1517-29.
  • cells can be cultured in the presence of other types of human feeder cells including, but not limited to, muscle cells, skin, fallopian tube epithelium, glandular endometrium, stromal endometrium, stroma of the medulla and foreskin, fetals and adults.
  • a suitable culture medium may comprise DMEM with 20% poor serum replacement and optional supplementation with non-amino acids. essentials, beta-mercaptoethanol and L-glutamine.
  • Other base media include G2.2, S2 (Scandanavian-2), and the like.
  • the described lines can be grown under conditions free of feeder cells, optionally in the presence of one or more growth factors that replace the feeder cells.
  • FGF fibroblast growth factor
  • FGFa or FGF1 acidic FGF
  • basic FGF or FGFb or FGF2
  • FGFb basic FGF
  • the amount of FGF can vary and a person skilled in the art can determine the amount required for the derivation and / or culture in an undifferentiated state. Suitable ranges include 1 - 1000 ng / mL, 1 - 100 ng / mL, 1 - 15 ng / mL, 1 - 10 ng / mL and 1 - 5 ng / mL.
  • Human FGF is preferred in some embodiments.
  • the CMEh can be propagated in culture indefinitely with regular passes, optionally on fresh feeder cells.
  • the derivation and propagation methods provided herein in some cases use feeder cells such as human feeder cells, and do not require the use of animal serum. Therefore, the probability of cross-species contamination using these methods is low to non-existent. Consequently, the CMEh lines and their compositions can be further characterized by the absence of animal pathogens and animal cells or cellular by-products.
  • the invention provides the use of CMEh lines generated within months or years of their derivation. Therefore, the CMEh lines can be stored indefinitely such as by cryopreservation.
  • the methods to cryopreserve CME are known in the art.
  • Cell freezing can be carried out using methods that include, but are not limited to, conventional slow freezing methods using dimethylsulfoxide (DMSO, preferably 10%) as a cryopreservation agent (as described by Bongso et al. Hum. Reprod. 9 (11): 2110-2117, 1994), vitrification methods (as described by Reubinoff et al. Hum. Reprod. 16 (10): 2187-2194, 2001), as well as other methods, such as described by Ji et al.
  • DMSO dimethylsulfoxide
  • CMEh lines can be used before cryopreservation, and directly from the culture.
  • the invention is not limited in this way.
  • the CMEh lines can be used for both therapeutic and research purposes.
  • the lines can be differentiated into one or more lineages.
  • the CMEh itself and / or its progeny can be used therapeutically.
  • the CMEh and / or their progeny can be used in vitro for various purposes, including the detection and identification of self-renewal factors and differentiation factors, and to test various factors, including supposed therapeutic candidate compounds.
  • the invention contemplates methods for differentiating the CMEh lines in one or more particular lineages including, but not limited to, endothelial cells, neurons, hematopoietic cells, cardiomyocytes, skeletal muscle cells, hepatocytes, insulin producing cells, glial progenitor cells. , osteoblasts, gametes and renal cells.
  • the CMEh lines can be used to regenerate a specific cell lineage (s), tissue or organ.
  • the invention also encompasses the resulting differentiated progeny that includes bone, cartilage, smooth muscle, heart muscle, skeletal muscle, kidney, striated muscle and hematopoietic cells (mesodermal lineages), liver, pancreas, thyroid gland, primitive intestine and respiratory epithelium (lineages endodermal), skin, pigment cells such as melanocytes, neurons, glia cells, hair follicles and dental outbreaks (ectodermal lineages) and their uses.
  • differentiation conditions are conditions that induce the CMEh to differ in one or more lineages. These conditions may vary according to the desired lineage. In general, you are Conditions may include the absence of feeder cells used to maintain the CMEh in an undifferentiated form, changes in the seeding density of the cells, and / or the introduction of one or more growth factors and / or other feeder cells that stimulate the differentiation in particular lineages.
  • Growth factors that can induce the differentiation of CMEh in particular lineages include, but are not limited to, retinoic acid, epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), fibroblast growth factor (FGF) , steroid hormones (e.g., glucocorticoids, vitamin A, thyroid hormone, androgens, estrogens and the like), activin-A (mesoderm), transforming growth factor beta 1 (TGF- / J1) (mesoderm), hepatocyte growth factor (HGF), and nerve growth factor (NGF).
  • retinoic acid retinoic acid
  • EGF epidermal growth factor
  • BMP4 bone morphogenetic protein 4
  • FGF fibroblast growth factor
  • steroid hormones e.g., glucocorticoids, vitamin A, thyroid hormone, androgens, estrogens and the like
  • activin-A meoderm
  • TGF- / J1 transforming growth factor beta 1
  • HGF hepatocyte growth factor
  • Differentiation in cardiac muscle can be induced using retinoic acid, 5-azacitidine and ascorbic acid.
  • Differentiation in hematopoietic lineages can be induced using bone marrow stromal cells, as described in US Pat. No. 6,613,568 and / or early-acting hematopoietic factors, such as kit ligand, IL-11, VEGF, Flk2 / Flt3 ligand and the like.
  • Differentiation in neuronal lineage can be induced using EGF and FGFb as described in the published US patent application. number 20050260747.
  • the CMEh lines can be used in the practice of transplants in the treatment (including prevention) of several states that affect one or more lineages.
  • Examples of such conditions include, but are not limited to, Parkinson's disease (dopaminergic neurons), Alzheimer's disease (neuronal precursors), Huntington's disease (GABAergic neurons), blood disorders such as leukemia, lymphoma, myeloma and anemia (hematopoietic cells), side effects of radiation, for example, in patients with transplantation (hematopoietic precursors), myocardial infarction, ischemic heart tissue or heart failure (partially or totally differentiated cardiomyocytes), muscular dystrophy (skeletal muscle cells), cirrhosis or liver failure (hepatocytes), chronic hepatitis (hepatocytes), diabetes including type I diabetes (insulin producing cells such as islet cells), ischemic brain injury (neurons), spinal cord injury (progenitor cells of the glia and motor neurons), amyotrophic lateral sclerosis (
  • the literature documenting the differentiation of embryonic stem cells in these various lineages includes Bjorklund et al., 2002, PNAS USA 99: 2344-2349 (dopaminergic neurons), West and Daley, 2004, Curr Opin CeII Biol 16: 688-692 ; US Pat. No. 6,534,052 B1; Kehat and Gepstein, 2003, Heart Fail Rev 8: 229-236; Nir et al., 2003, Cardiovasc Res 58: 313-323; U.S. patents Nos. 6,613,568 and 6,833,269.
  • the invention comprises transplants of differentiated cells and / or undifferentiated or partially differentiated embryonic stem cells.
  • the CMEh and / or its differentiated progeny can be introduced into a subject locally or systemically by various methods and routes.
  • Local administration includes direct injection into particular sites in the body, including normal and abnormal organs and tissues. Such local administration can be done by direct needle injection.
  • Systemic administration encompasses parenteral (eg, intravenous, intramuscular, subcutaneous, intraperitoneal, intratumoral, intrathecal, etc.) and non parenteral administration routes.
  • the CMEh lines and / or their differentiated progeny can be provided in pharmaceutical preparations. Such preparations are suitable for administration in vivo and, therefore, are minimally sterile and physiologically acceptable to the recipient. These preparations may generally comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable vehicle means a non-toxic material that does not interfere with the effectiveness of the cells and / or other agents administered. Pharmaceutically acceptable vehicles include diluents, fillers, salts, buffers, stabilizers, preservatives, solubilizers and other materials that are well known in the art.
  • Pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic and the like.
  • pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.
  • Pharmaceutical preparations may also contain other therapeutic agents.
  • the invention also encompasses pharmaceutical preparations that are formulated for local administration, such as implants. Examples of bioerodible implants are described in the international PCT application number PCT / US / 03307 (publication number WO 95/24929).
  • the invention also contemplates the ability to transduce embryonic stem cells and / or their differentiated progeny with particular nucleic acids, thus giving rise to genetically modified stem cells and progeny. If they are intended for transplantation, these cells can be used, for example, to generate particular factors or to complement particular mutations in the recipient.
  • the transduction of the CMEh lines is also taught in the US patent application publication. Number 20050079616.
  • embryonic stem cell transduction refers to the process of transferring exogenous genetic material into an embryonic stem cell.
  • transduction transfection
  • transformation refers to the process of transferring exogenous genetic material to a cell.
  • exogenous genetic material refers to natural or synthetic nucleic acids or oligonucleotides, which are introduced into cells.
  • the exogenous genetic material may be a copy of it that is naturally present in the cells, or may not be found naturally in cells. Normally, it is at least a part of a naturally occurring gene that has been placed under the operational control of a promoter in a vector construct.
  • nucleic acids can be introduced into cells. Such techniques include the transfection of nucleic acid precipitates - CaPO 4 , the transfection of nucleic acids associated with DEAE, the transfection with a retrovirus that includes the nucleic acid of interest, liposome-mediated transfection and the like. For certain uses, it is preferred to select the target nucleic acid for particular cells.
  • a vehicle used to deliver a nucleic acid according to the invention in a cell for example, a retrovirus, or another virus; a liposome
  • a target selection molecule attached to it.
  • a molecule such as an antibody specific for a surface membrane protein in a target cell or a ligand for a receptor in the target cell, can be attached or incorporated into the nucleic acid delivery vehicle.
  • proteins can be incorporated that bind to a surface membrane protein associated with endocytosis in the liposome formulation to select as a target and / or facilitate the uptake.
  • proteins include proteins or fragments of the same tropics for a particular cell type, antibodies for proteins that undergo intemalization, proteins that confer intracellular localization and / or that enhance intracellular half-life and the like.
  • Polymeric administration systems have also been successfully used to administer nucleic acids in the cells, as is known to those skilled in the art.
  • Retroviruses deficient in replication can direct the synthesis of all virion proteins, but cannot form infectious particles. Consequently, these genetically altered retroviral vectors have a general utility for the transduction of genes of high efficiency in cells in culture, and a utility specific for use in the method of the present invention. Retroviruses have been widely used to transfer genetic material to cells.
  • a preferred retroviral expression vector includes an exogenous promoter element to control the transcription of the inserted exogenous gene.
  • exogenous promoters include both constitutive and inducible promoters.
  • the main advantage of the use of retroviruses is that the virus effectively inserts a single copy of the gene that codes for the therapeutic agent in the genome of the host cell, thus allowing the exogenous genetic material to pass to the progeny of the cell when it is divided.
  • gene promoter sequences in the LTR region enhance the expression of a coding sequence inserted in a variety of cell types.
  • the main disadvantages of using a retrovirus expression vector are (1) the insertional mutagenesis, that is, the insertion of the therapeutic gene in an undesired position in the genome of the target cell, which, for example, leads to lack of regulation of cell growth and (2) the need for proliferation of the target cell so that the therapeutic gene carried by the vector is integrated into the target genome.
  • Still another viral candidate useful as an expression vector for cell transformation is adenovirus, a double-stranded DNA virus.
  • the adenovirus genome can be adapted for use as an expression vector for gene transduction, that is, by eliminating The genetic information that controls the production of the virus itself. Since the adenovirus normally works in an extrachromosomal way, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis. On the other hand, the transformation with adenovirus of a target cell may not result in a stable transduction.
  • adenovirus sequences confer intrachromosomal integration specificity for vehicle sequences and, therefore, result in a stable transduction of exogenous genetic material. Therefore, as will be apparent to one skilled in the art, a variety of vectors suitable for transferring exogenous genetic material into the cells are available.
  • the selection of an appropriate vector to administer a therapeutic agent for a particular state that can be treated with gene replacement therapy and the optimization of the conditions for the insertion of the selected expression vector in the cell are within the scope of an expert. usual in the technique, without the need for excessive experimentation.
  • the promoter characteristically has a specific nucleotide sequence necessary to initiate transcription.
  • the exogenous genetic material also includes additional sequences (ie enhancers (enhancers)) required to obtain the desired gene transcription activity.
  • an "enhancer” is simply any non-translated DNA sequence that works with the coding sequence (in cis) to change the level of basal transcription dictated by the promoter.
  • the exogenous genetic material is introduced into the genome immediately in the 3 'direction from the promoter, so that the promoter and the coding sequence are operatively linked to allow transcription of the coding sequence.
  • the constitutive promoters that occur in nature control the expression of the essential functions of the cell. As a result, a gene under the control of a constitutive promoter is expressed in all cell growth conditions.
  • Example constitutive promoters include promoters of the following genes that code for certain functions constitutive or "housekeeping": hypoxanthine phosphoribosyl tranf ⁇ rase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., Proc. Nati. Acad.
  • any of the constitutive promoters named above can be used to control the transcription of a heterologous gene insert.
  • inducible promoters include response elements (ER) that stimulate transcription when their induction factors are joined.
  • ER response elements
  • ER response elements
  • ER for serum factors, steroid hormones, retinoic acid and cyclic AMP.
  • Promoters containing a particular ER can be chosen in order to obtain an inducible response and, in some cases, the ER itself can bind to a different promoter, thereby conferring induction capacity on the recombinant gene.
  • the expression vector preferably includes a selection gel, for example, a neomycin resistance gene, to facilitate the selection of cells that have been transfected or transduced with the expression vector.
  • the cells are transfected with two or more expression vectors, at least one vector containing the gene (s) encoding the therapeutic agent (s), containing the other vector. a selection gene.
  • the selection of a suitable promoter, enhancer, selection gene and / or signal sequence is considered to be within the scope of a person skilled in the art without excessive experimentation.
  • the selection and optimization of a particular expression vector to express a specific gene product in a cell is carried out by obtaining the gene, preferably with one or more appropriate control regions (for example, promoter, insertion sequence); Ia preparation of a vector construct comprising the vector in which the gene is inserted; The transfection or transduction of cells in in vitro culture with the vector construct; and the determination of whether the gene product is present in the cells.
  • appropriate control regions for example, promoter, insertion sequence
  • Table 1 only represents examples of genes that can be supplied according to the methods of the invention.
  • useful genes substitute or complement the function, including genes that code for missing enzymes such as adenosine deaminase (ADA) that has been used in clinical trials to treat ADA deficiency and cofactors such as insulin and coagulation factor VIII.
  • ADA adenosine deaminase
  • the invention further contemplates the selection of various compounds for their effects on the CMEhs provided herein.
  • the compounds can be selected for their ability to maintain the CMEh lines in an undifferentiated state, or to induce the differentiation of the CMEh, or otherwise modify the cells. Some aspects of the selection may be directed to test the therapeutic efficacy of candidate compounds. Depending on the particular compounds being selected, test readings will vary. For example, in some tests, the reading will be the maintenance and / or increase in the number of CMEh while in others the reading will be the production of differentiated progeny (optionally with a concomitant decrease in the number of CMEh). The differentiation of the CMEh can also be followed through changes in the expression profiles.
  • the differentiation of the CMEh can be identified by the regulation by decrease of the expression of SSEA-3 and SSEA-4 and the regulation by increase of the expression of SSEA-1.
  • Still other tests may include a reading of cell viability or alternatively cell death. Such tests can then provide in vitro readings that potentially correlate with the toxicity and / or efficacy that the test compounds would show in human subjects. Therefore, the effect of the agent on the CMEh line or its differentiated progeny in vitro is a form of marker or substitute reading on how the live agent will work.
  • the lines can be used additionally as a model system in which to develop a personalized therapeutic regimen for a patient that can be genetically similar or histocompatible with the line.
  • the first two human embryonic stem cell lines (VAL-1 and VAL-2) have been derived in Spain with long-term cryopreserved embryos in animal-free conditions. All frozen embryos for> 5 years were donated after informed consent for stem cell bypass, according to Spanish law 45/2003 of November 21, 2003. Forty human embryos that had been cryopreserved on day 2 of development were thawed. > 5 years. Six embryos did not survive this process: 5 degenerated and the area of 1 embryo fractured. A total of 15 of 34 embryos (44.1%) were stopped during the initial stages of development: 12 growth was stopped on the first day (35.3%) and 3 (8.8%) subsequently. Additionally, 3 pseudoblasts were formed (8.8%).
  • blastocysts In total, 16 blastocysts (47.1%) were obtained and classified according to Gardner et al. Fertile Steril 1998 69: 84-88. Of these, 11 blastocysts (68.7%) had a degree of internal cell mass (MCI) of either A or B.
  • MCI degree of internal cell mass
  • placental fibroblast lines A complete description of the production of placental fibroblast lines has been described by Genbacev et al. Fertile Steril 2005 83: 1517-1529.
  • Human placental fibroblasts obtained from human placentas of early gestation support the propagation of established CMEh lines.
  • placental fibroblasts were comparable to mouse fibroblasts as feeder cells for CMEh.
  • a line of qualified placental fibroblasts was used as a feeder layer for the derivation of new CMEh.
  • the zona pellucida with Tyrode acid solution was removed as described in Genbacev et al., And the blastocysts (see Figures 1A and D) were plated on feeder cells formed from irradiated human placental fibroblasts in Defined medium: 79% of Dulbecco's minimum essential medium (DMEM) deficient (Gibco / BRL, Paisley, United Kingdom), 20% deficient recombinant serum (SR) (Gibco / BRL), 1 mmol / L glutamine (Gibco / BRL ), ⁇ -mercaptoethanol 0.1 mmol / L (Sigma, St.
  • DMEM Dulbecco's minimum essential medium
  • SR deficient recombinant serum
  • Sibco / BRL 1 mmol / L glutamine
  • ⁇ -mercaptoethanol 0.1 mmol / L
  • Non-essential amino acid reserve (Gibco / BRL), which contains 12 ng / mL basic human fibroblast growth factor (Invitrogen; Life Technologies, Carlsbad, CA).
  • the referral process was carried out according to GMP.
  • VAL-1 and VAL-2 have been cryopreserved and thawed successfully using the conventional slow freezing method with 10% dimethylsulfoxide as a cryoprotectant, as described by Bongso et al. Hum. Play 11: 2110-2117, 1994.
  • the colonies of VAL-1 and VAL-2 had a larger surface area, appeared thinner and flatter, and had straight defined boundaries, providing the colonies with angular or circular edges (see Figures 1 C and F) .
  • the individual CMEh on human feeder cells were small and round, with prominent nucleoli, a typical characteristic of these cells.
  • the karyotype of the cells was obtained each time the colonies were divided.
  • HESCs were incubated in hES, supplemented with colcemid 0.2 .mu.g / ml (ROCHE, stock solution 10 mg / ml) at 37 0 C for 30 minutes, and subsequently washed three times with 2 mi PBS + Ca of + Mg
  • a minimum of 20 colonies on PBS were mechanically dissected from Ia feeder layer, they were collected in 2 ml of 1x trypsin-EDTA and incubated at 37 0 C for 5 minutes. The final mixture of cells was pipetted several times at the end of the incubation, in order to guarantee total disintegration in individual cells. Trypsin activity was stopped with 4 ml of hES medium and centrifuged at 1800 rpm for 10 minutes.
  • Cytogenetic analyzes of at least 20 metaphase distributions and five band karyotypes were evaluated to determine chromosomal redispositions using the GTG band method by two qualified geneticists at Prenatal Genetics (Barcelona, Spain). Each analysis showed that both cell lines maintained a normal 46, XX karyotype (see Figures 2A and F).
  • TRA-1-60 and TRA-1-81 were exposed to specific primary antibodies to tumor rejection antigens TRA-1-60 and TRA-1-81 (generously provided by Peter Andrews, Sheffield University), and phase 4 specific embryonic antigen (SSEA-4) , (Chem ⁇ con, Temecula, CA).
  • SSEA-4 phase 4 specific embryonic antigen
  • the alkaline phosphatase activity was demonstrated by means of a Blue / Red vector substrate kit (Vector Laboratories, Burlingame, CA). Immunolocation studies showed that VAL-1 and VAL-2 expressed SSEA-4 (Chemicon; Temecula, CA) (see Figures 2B and G), TRA-1-60 (see Figures 2C and H) and TRA-1 -81 (see Figures 2D and I).
  • RNA from VAL-1 and VAL-2 grade A colonies was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions for small-scale cell quantities, and the RNA concentration was evaluated using a spectrophotometer (BioRad) .
  • Total RNA (1 ⁇ g) of each sample was used for a first strand cDNA synthesis using the Advantage RT PCR kit (BD Biosciences) following the manufacturer's protocol.
  • PCR primers were designed using the network-based software available for free (Primer3, Genefisher).
  • PCR reactions were carried out using 1 ⁇ g of total cDNA as a template, as follows: denaturation at 94 0 C for 4 minutes, and cycles 40 times at 94 0 C for 1 minute, 55 0 C for 1 minute and 72 0 C for 1 minute. A final extension was performed at 72 0 C for 10 minutes after the cycles.
  • the PCR products were resolved in 2% agarose gels, stained with ethidium bromide, and visualized on a transilluminator (BioRad). These RT-PCR studies showed that both cell lines were positive for Oct-4, Rex-1, Crypto, Thy-1, and Nanog and were negative for classic differentiation markers: Matni, Amylase, and Dbh.
  • telomerase activity of VAL-1 and VAL-2 was analyzed using a TRAPEZE ® (Chemicon) telomerase detection kit followed by staining with SYBR ® (Molecular Probes, " Eugene, OR). Briefly, colonies were collected (50- 100) washed once with PBS free of Ca ++ and Mg ++ and immediately resuspended in lysis buffer After ice treatment and centrifugation, the samples were subjected to a PCR reaction following the manufacturer's instructions.
  • PCR were tested on a polyacrylamide gel (15% TBE, BioRad) under non-denaturing conditions, and the amplified fragments were stained with SYBR green for visualization in a transilluminator.
  • the negative controls were obtained by thermal inactivation of the
  • the final product contained a scale of amplification products with increments of 6 bp starting at 50 bp (see Figure 3)
  • the band of 36 bp corresponds to the internal control of the reaction in polymerase chain and The 50 bp band, at the telomerase activity that increases in 6 bp bands in the immortal cells.
  • VAL-1 and VAL-2 were immunologically identical for the markers of HLA, A2, A23, B44, B40, CW4, CW5, DR7, DR15, DQ2 and DQ6. This level of histocompatibility indicates that the lines can be used together in a therapeutic regimen with little or no risk of immune rejection. However, the lines are not genetically identical, as shown in Figures 4, 5 and 6.
  • VAL-1 and VAL-2 were also evaluated to determine their spontaneous differentiation profiles. Colonies were dissociated by collagenase IV treatment for 5 minutes at 37 0 C and then grown in suspension culture plates 6 wells of poor binding.
  • the differentiation medium consisted of 80% DMEM, 20% FBS (Hyclone), 1 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, and MEM 1 mM non-essential amino acids. After 4 days in suspension, the embryoid bodies were transferred in culture chambers coated with poly-L-ornithine and cultured for an additional 10-14 days. Cultures were fixed with 4% paraformaldehyde for 20 minutes before immunolocation analyzes.
  • the embryoid bodies were incubated with, for example, mouse anti- ⁇ -fetoprotein (ascites) (diluted 1: 500; Sigma), anti- ⁇ - mouse tubulin III (ascites) (diluted 1: 1, 000; Sigma), or anti-actin of the mouse smooth muscle (10.7 ⁇ g / mL; Dako).
  • Negative controls included the omission of the primary antibodies and the incubation with a non-specific IgG.
  • VAL-1 showed the ability to differentiate spontaneously in cells similar to myocardiocytes ( Figures 7A-D), and both VAL-1 and VAL-2 showed the ability to differentiate spontaneously in cells similar to neurons (Figure 8B), and similar cells to the muscle ( Figure 8C).
  • the gene expression profiles of the spontaneously differentiated progeny of the lines were also analyzed. As shown in Table 2, the lines can spontaneously express genes associated with endoderm, mesoderm and ectoderm lineages. Table 2. Gene expression profile of spontaneous differentiation.

Abstract

The invention provides novel human embryo stem-cell lines. The invention also provides human embryo stem-cell lines that are genetically related and are immunologically identical. The invention also provides methods for deriving, propagating and utilizing such lines, optionally under animal-free conditions.

Description

LÍNEAS DE CÉLULAS MADRE EMBRIONARIAS HUMANAS Y MÉTODOS PARA USAR LAS MISMASLINES OF HUMAN EMBRYOUS MOTHER CELLS AND METHODS TO USE THE SAME
Campo de Ia invención La invención se refiere a líneas celulares embrionarias humanas y a métodos para usar las mismas.Field of the invention The invention relates to human embryonic cell lines and methods for using them.
Antecedentes de Ia invenciónBackground of the invention
Las células madre embrionarias humanas (CMEh) tienen Ia capacidad de diferenciarse en varios linajes celulares diferentes in vitro e in vivo incluyendo, pero sin limitarse a, melanocitos, células hematopoyéticas, hepatocitos, células renales, células del músculo esquelético, neuronas dopaminérgicas, células de Ia glía, cardiomiocitos, células endoteliales y osteoblastos. Las células CMEh son terapéuticamente atractivas debido a su pluripotencia. El uso de tales células y su progenie diferenciada se contemplan para el tratamiento de diversos estados. Sin embargo, hay algunos inconvenientes actuales a las líneas de CMEh existentes actualmente.Human embryonic stem cells (CMEh) have the ability to differentiate into several different cell lineages in vitro and in vivo including, but not limited to, melanocytes, hematopoietic cells, hepatocytes, renal cells, skeletal muscle cells, dopaminergic neurons, Ia glia, cardiomyocytes, endothelial cells and osteoblasts. CMEh cells are therapeutically attractive due to their pluripotence. The use of such cells and their differentiated progeny are contemplated for the treatment of various conditions. However, there are some current drawbacks to the existing CMEh lines currently.
En primer lugar, el transplante de CMEh (y su progenie diferenciada) en sujetos humanos requerirá, en Ia mayoría de los casos, histocompatibilidad entre las CMEh y los receptores humanos de tales células. Dado el nivel de diversidad en los antígenos de histocompatibilidad humanos, se ha calculado que se necesitaría generar 150.000 líneas.In the first place, the transplantation of CMEh (and its differentiated progeny) in human subjects will require, in the majority of cases, histocompatibility between the CMEh and the human receptors of such cells. Given the level of diversity in human histocompatibility antigens, it has been calculated that 150,000 lines would need to be generated.
En segundo lugar, el tratamiento de los sujetos humanos con líneas de CMEh (y su progenie diferenciada), incluyendo en Ia práctica de transplantes, también requerirá casi con toda seguridad que las líneas se generen en ausencia de productos animales (es decir, no humanos). El primer informe de CMEh utilizó células alimentadoras (feeder) de fibroblasto de ratón para derivar y cultivar las células. Véanse las patentes de los EE.UU. 5.843.780 y 6.200.806. Tal enfoque puede dar como resultado Ia transmisión de patógenos de ratón en sujetos humanos.Second, the treatment of human subjects with CMEh lines (and their differentiated progeny), including in the practice of transplants, will also almost certainly require that the lines be generated in the absence of animal products (that is, non-human ). The first CMEh report used mouse fibroblast feeder cells to derive and cultivate the cells. See US patents. 5,843,780 and 6,200,806. Such an approach can result in the transmission of mouse pathogens in human subjects.
En tercer lugar, aunque se ha notificado que existen en Ia actualidad del orden de 75 - 100 líneas de CMEh, sólo se ha confirmado que algunas de ellas tienen auténticas propiedades de células madre, incluyendo autorrenovación (como se pone de manifiesto por el cultivo a largo plazo) y diferenciación de linaje pluripotente.Thirdly, although it has been reported that there are currently around 75-100 CMEh lines, it has only been confirmed that some of them they have authentic properties of stem cells, including self-renewal (as evidenced by long-term culture) and differentiation of pluripotent lineage.
Existe una necesidad de más líneas de CMEh con el fin de superar estos temas.There is a need for more CMEh lines in order to overcome these issues.
Sumario de Ia invenciónSummary of the invention
La invención parte de Ia premisa, en parte, de Ia derivación y caracterización de líneas de CMEh a partir de embriones humanos crioconservados a largo plazo. Las líneas se derivaron en un medio definido sin suero y sin células alimentadoras animales (es decir, condiciones libres de animales). Dos ejemplos de estas líneas se designan VAL-1 y VAL-2. Estas líneas se derivaron de embriones "hermanos" (es decir, embriones que tienen origen idéntico) y son inmunológicamente idénticos. En consecuencia, estas líneas pueden utilizarse juntas, por ejemplo, en una práctica terapéutica, debido a su histocompatibilidad.The invention starts from the premise, in part, of the derivation and characterization of CMEh lines from long-term cryopreserved human embryos. The lines were derived in a defined medium without serum and without animal feeder cells (i.e., animal-free conditions). Two examples of these lines are designated VAL-1 and VAL-2. These lines were derived from "brother" embryos (ie, embryos that have identical origins) and are immunologically identical. Consequently, these lines can be used together, for example, in a therapeutic practice, due to their histocompatibility.
Por tanto, en un aspecto, Ia invención proporciona una población aislada de células madre embrionaria humanas en Ia que dicha población de células (1) tiene un cariotipo normal tras al menos 85 pases; (2) expresa el antígeno embrionario específico de fase (SSEA) 4, el antígeno de rechazo tumoral (TRA)-I -60 y TRA-1-81; (3) es positiva para fosfatasa alcalina; (4) expresa Oct- 4, Rex-1 , Cripto, Thy-1 y Nanog; (5) es negativa para Matni , Amylase y Dbh; y (6) tiene actividad telomerasa. Estas características (o propiedades) se mantienen preferiblemente durante el largo plazo (por ejemplo, tras al menos 85 pases).Therefore, in one aspect, the invention provides an isolated population of human embryonic stem cells in which said population of cells (1) has a normal karyotype after at least 85 passes; (2) expresses the phase-specific embryonic antigen (SSEA) 4, the tumor rejection antigen (TRA) -I-60 and TRA-1-81; (3) is positive for alkaline phosphatase; (4) expresses Oct-4, Rex-1, Crypto, Thy-1 and Nanog; (5) is negative for Matni, Amylase and Dbh; and (6) has telomerase activity. These characteristics (or properties) are preferably maintained for the long term (for example, after at least 85 passes).
En otros aspectos, Ia invención proporciona una población aislada de células madre embrionarias humanas, en Ia que dicha población de células tiene las características de Ia línea de células madre embrionarias VAL-1 o VAL-2. Todavía en otros aspectos, Ia invención proporciona una población aislada de células madre embrionarias, en Ia que dicha población de células es Ia línea de células madre embrionarias VAL-1 o VAL-2, y Ia progenie de Ia misma. La progenie puede ser progenie diferenciada o no diferenciada incluyendo, pero sin limitarse a, progenie cardiaca, neuronal, muscular y hematopoyética.In other aspects, the invention provides an isolated population of human embryonic stem cells, in which said population of cells has the characteristics of the VAL-1 or VAL-2 embryonic stem cell line. In still other aspects, the invention provides an isolated population of embryonic stem cells, in which said population of cells is the VAL-1 or VAL-2 embryonic stem cell line, and the progeny of the same. The progeny can be differentiated or undifferentiated progeny including, but not limited to, cardiac, neuronal, muscular and hematopoietic progeny.
En algunas realizaciones, Ia población comprende células modificadas genéticamente.In some embodiments, the population comprises genetically modified cells.
En algunas realizaciones, Ia población se somete a pases al menos 50 veces, al menos 75 veces, al menos 100 veces, o más. En una realización, Ia población se somete a pases al menos 85 veces o al menos 120 veces.In some embodiments, the population is subjected to passes at least 50 times, at least 75 times, at least 100 times, or more. In one embodiment, the population is subjected to passes at least 85 times or at least 120 times.
En algunas realizaciones, Ia población se ha crioconservado y opcionalmente descongelado y sometido a pases, sin cambios en Ia expresión del marcador, el cariotipo o Ia actividad telomerasa. La población puede crioconservarse durante más de 1 año.In some embodiments, the population has been cryopreserved and optionally thawed and subjected to passes, without changes in the expression of the marker, the karyotype or the telomerase activity. The population can be cryopreserved for more than 1 year.
Las "poblaciones" de células madre embrionarias humanas mencionadas en el presente documento también se denominan "líneas" de células madre embrionarias humanas.The "populations" of human embryonic stem cells mentioned herein are also called "lines" of human embryonic stem cells.
En otro aspecto, Ia invención proporciona un par de poblaciones (o líneas) aisladas de células madre embrionarias humanas que expresan los marcadores de HLA (antígeno leucocitario humano) A2, A23, B44, B40, CW4, CW5, DR7, DR15, DQ2 y DQ6. La invención proporciona composiciones y métodos para usar el par de poblaciones de células madre embrionarias humanas (y/o su progenie diferenciada). En un aspecto, el par de líneas (y/o su progenie diferenciada) se utilizan en sujetos humanos que tienen uno o más marcadores de los HLA mencionados anteriormente. Las poblaciones (y/o su progenie diferenciada) pueden introducirse en los sujetos al mismo tiempo o en momentos diferentes. Los métodos pueden comprender además el uso de diferentes poblaciones diferenciadas de las líneas en el mismo sujeto. Como ejemplo, un sujeto puede recibir células hematopoyéticas derivas de VAL-1 y queratinocítos derivados de VAL-2 al mismo tiempo o en momentos diferentes.In another aspect, the invention provides a pair of populations (or lines) isolated from human embryonic stem cells that express the markers of HLA (human leukocyte antigen) A2, A23, B44, B40, CW4, CW5, DR7, DR15, DQ2 and DQ6. The invention provides compositions and methods for using the pair of human embryonic stem cell populations (and / or their differentiated progeny). In one aspect, the pair of lines (and / or their differentiated progeny) are used in human subjects who have one or more of the HLA markers mentioned above. The populations (and / or their differentiated progeny) can be introduced into the subjects at the same time or at different times. The methods may further comprise the use of different populations differentiated from the lines in the same subject. As an example, a subject can receive hematopoietic cells derived from VAL-1 and keratinocytes derived from VAL-2 at the same time or at different times.
Todavía en otro aspecto, Ia invención proporciona composiciones que comprenden una o más células madre embrionarias humanas de cualquiera de las poblaciones anteriores de células madre embrionarias humanas. La composición puede ser una preparación farmacéutica, pero no se limita a ella. - A -In another aspect, the invention provides compositions comprising one or more human embryonic stem cells of any of the previous populations of human embryonic stem cells. The composition may be a pharmaceutical preparation, but it is not limited thereto. - TO -
Todavía en otro aspecto, Ia invención proporciona un cultivo que comprende cualquiera de las poblaciones anteriores de células madre. El cultivo puede comprender células alimentadoras, preferiblemente células alimentadoras humanas tales como, pero sin limitarse a, células alimentadoras placentarias humanas. En ciertas realizaciones, las células alimentadoras se inactivan mitóticamente tal como, por ejemplo, por irradiación. El cultivo puede ser un cultivo libre de suero. El cultivo puede comprender factor de crecimiento de fibroblastos (FGF) tal como, pero sin limitarse al FGF básico (FGFb). En algunas realizaciones, el cultivo puede estar libre de células alimentadoras. En las realizaciones preferidas, el cultivo está libre de productos animales (es decir, no humanos). El cultivo puede comprender células madre embrionarias humanas y Ia progenie diferenciada de las mismas.In yet another aspect, the invention provides a culture comprising any of the previous populations of stem cells. The culture may comprise feeder cells, preferably human feeder cells such as, but not limited to, human placental feeder cells. In certain embodiments, feeder cells are mitotically inactivated such as, for example, by irradiation. The culture may be a serum free culture. The culture may comprise fibroblast growth factor (FGF) such as, but not limited to the basic FGF (FGFb). In some embodiments, the culture may be free of feeder cells. In preferred embodiments, the culture is free of animal products (ie, non-human). The culture may comprise human embryonic stem cells and the differentiated progeny thereof.
Aún en otro aspecto, Ia invención proporciona un método para cultivar que incluye Ia propagación de poblaciones de células madre embrionarias humanas que comprende cultivar cualquiera de las poblaciones anteriores de células madre embrionarias humanas en un medio libre de suero y opcionalmente en presencia de células alimentadoras humanas. Las células alimentadoras humanas pueden ser células alimentadoras placentarias humanas. Las células alimentadoras pueden inactivarse mitóticamente, por ejemplo, por irradiación. El medio puede comprender FGF tal como, pero sin limitarse al, FGFb. En algunas realizaciones, el FGFb está presente en una cantidad de aproximadamente 1 - 15 ng/mL o 1-10 ng/mL.In another aspect, the invention provides a method for cultivation that includes the propagation of human embryonic stem cell populations comprising culturing any of the previous populations of human embryonic stem cells in a serum-free medium and optionally in the presence of human feeder cells. . Human feeder cells can be human placental feeder cells. Feeder cells can be inactivated mitotically, for example, by irradiation. The medium may comprise FGF such as, but not limited to, FGFb. In some embodiments, the FGFb is present in an amount of about 1-15 ng / mL or 1-10 ng / mL.
Todavía en otro aspecto, Ia invención proporciona un método para diferenciar ¡n vitro una población de células madre embrionarias humanas que comprende exponer cualquiera de las poblaciones anteriores de células madre embrionarias humanas a condiciones de diferenciación durante un tiempo suficiente para permitir Ia diferenciación de Ia población de células madre embrionarias humanas en células diferenciadas. Dependiendo de Ia realización, las condiciones de diferenciación pueden comprender células alimentadoras o pueden estar libres de células alimentadoras. En algunas realizaciones, las condiciones de diferenciación comprenden uno o más factores seleccionados del grupo que consiste en ácido retinoico, factor de crecimiento epidérmico (EGF), proteína morfogenética ósea 4 (BMP4), factor de crecimiento de fibroblastos (FGF), hormonas esteroideas, activina-A, factor de crecimiento transformante beta 1 (TGF-/J1), factor de crecimiento de hepatocitos (HGF) y factor de crecimiento nervioso (NGF). El método puede comprender además introducir las células diferenciadas en un sujeto tal como, pero sin limitarse a, un sujeto humano.In yet another aspect, the invention provides a method for differentiating in vitro a population of human embryonic stem cells comprising exposing any of the previous populations of human embryonic stem cells to differentiation conditions for a time sufficient to allow the differentiation of the population of human embryonic stem cells in differentiated cells. Depending on the embodiment, the differentiation conditions may comprise feeder cells or may be free from feeder cells. In some embodiments, the differentiation conditions comprise one or more factors selected from the group consisting of retinoic acid, a factor of epidermal growth (EGF), bone morphogenetic protein 4 (BMP4), fibroblast growth factor (FGF), steroid hormones, activin-A, transforming growth factor beta 1 (TGF- / J1), hepatocyte growth factor (HGF ) and nerve growth factor (NGF). The method may further comprise introducing the differentiated cells into a subject such as, but not limited to, a human subject.
Todavía en otro aspecto, Ia invención proporciona un método para diferenciar una población de células madre embrionarias humanas que comprende introducir cualquiera de las poblaciones anteriores de células madre embrionarias humanas en un sujeto incluyendo, pero sin limitarse a, un sujeto humano. En algunas realizaciones, el sujeto tiene un estado que afecta al hígado, músculo, piel, cerebro, sistema nervioso, corazón, sistema circulatorio, sistema hematopoyético, páncreas o hueso. En algunas realizaciones, Ia población se introduce en el sujeto mediante administración local, tal como, pero sin limitarse a, Ia administración a un órgano o a un tejido. En algunas realizaciones, Ia población se introduce en el sujeto mediante administración sistémica, tal como, pero sin limitarse a, administración intravenosa. En algunas realizaciones, el método comprende además exponer Ia población a condiciones de diferenciación in vitro antes de Ia introducción en el sujeto. En algunas realizaciones, el sujeto expresa uno, dos, tres, cuatro, cinco, seis, siete, ocho o nueve marcadores de HLA seleccionados del grupo que consiste en A2, A23, B44, B40, CW4, CW5, DR7, CD15, DQ2 y DQ6. En algunas realizaciones, el sujeto expresa todos los marcadores anteriores. En algunas realizaciones, el sujeto recibe las células VAL-1 y VAL-2 o Ia progenie de las mismas.In another aspect, the invention provides a method for differentiating a population of human embryonic stem cells that comprises introducing any of the previous populations of human embryonic stem cells into a subject including, but not limited to, a human subject. In some embodiments, the subject has a state that affects the liver, muscle, skin, brain, nervous system, heart, circulatory system, hematopoietic system, pancreas or bone. In some embodiments, the population is introduced into the subject by local administration, such as, but not limited to, administration to an organ or tissue. In some embodiments, the population is introduced into the subject by systemic administration, such as, but not limited to, intravenous administration. In some embodiments, the method further comprises exposing the population to differentiation conditions in vitro before the introduction into the subject. In some embodiments, the subject expresses one, two, three, four, five, six, seven, eight or nine HLA markers selected from the group consisting of A2, A23, B44, B40, CW4, CW5, DR7, CD15, DQ2 and DQ6. In some embodiments, the subject expresses all the previous markers. In some embodiments, the subject receives the VAL-1 and VAL-2 cells or the progeny thereof.
Aún en otro aspecto, Ia invención proporciona un método para determinar Ia eficacia terapéutica o Ia citotoxicidad de un compuesto que comprende exponer cualquiera de las líneas anteriores de células madre embrionarias humanas a un compuesto (por ejemplo, poniendo en contacto Ia línea con el compuesto), y determinar un efecto del compuesto sobre Ia línea de células madre embrionarias humanas. La línea puede diferenciarse antes de ponerse en contacto con el compuesto. La diferenciación puede ser diferenciación hematopoyética o diferenciación neuronal, pero Ia invención se limita a ellas. El efecto del compuesto puede ser Ia muerte celular, el crecimiento celular (por ejemplo, aumento en el número de células), el aumento en el número de células madre embrionarias humanas no diferenciadas, Ia disminución en el número de células madre embrionarias humanas no diferenciadas (por ejemplo, debido a diferenciación), el aumento en el número de células de un linaje diferenciado particular, los cambios en los perfiles de expresión, y similares.In yet another aspect, the invention provides a method for determining the therapeutic efficacy or cytotoxicity of a compound comprising exposing any of the previous lines of human embryonic stem cells to a compound (for example, by contacting the line with the compound) , and determine an effect of the compound on the human embryonic stem cell line. The line can be differentiated before contacting the compound. The differentiation can be hematopoietic differentiation or neuronal differentiation, but the invention is limited to them. The effect of the compound can be cell death, cell growth (for example, increase in the number of cells), the increase in the number of undifferentiated human embryonic stem cells, the decrease in the number of undifferentiated human embryonic stem cells (for example, due to differentiation), the increase in the number of cells of a particular differentiated lineage, changes in expression profiles, and the like.
Estas y otras realizaciones de Ia invención se describirán en mayor detalle en el presente documento.These and other embodiments of the invention will be described in greater detail herein.
Cada una de las limitaciones de Ia invención puede englobar varias realizaciones de Ia invención. Por tanto, se prevé que puede estar incluida en cada uno de los aspectos de Ia invención cada una de las limitaciones de Ia invención que suponga cualquier elemento o combinación de elementos. Esta invención no está limitada en su aplicación a los detalles de construcción y a Ia disposición de los componentes expuestos en Ia siguiente descripción o ilustrados en los dibujos. La invención permite otras realizaciones y ponerse en práctica o llevarse a cabo de varias formas.Each of the limitations of the invention can encompass several embodiments of the invention. Therefore, it is envisioned that each of the limitations of the invention may be included in each of the aspects of the invention that any element or combination of elements entails. This invention is not limited in its application to the construction details and to the arrangement of the components set forth in the following description or illustrated in the drawings. The invention allows other embodiments and to be practiced or carried out in various ways.
La fraseología y Ia terminología utilizadas en el presente documento son con fines de descripción y no deben considerarse como limitantes. El uso de "que incluye", "que comprende" o "que tiene", "que contiene", "que supone" y variaciones de los mismos en el presente documento, se refieren a que engloban los puntos enumerados en ellos y equivalentes de los mismos, así como a puntos adicionales.The wording and terminology used in this document are for the purpose of description and should not be considered as limiting. The use of "that includes", "that includes" or "that has", "that contains", "that supposes" and variations of the same in the present document, refer to that they include the points listed in them and equivalents of the same, as well as additional points.
Breve descripción de las figurasBrief description of the figures
Las figuras 1A-F son una serie de fotografías que muestran las características morfológicas de Ia derivación de VAL-1 y VAL-2. Se descongelaron dos embriones diferentes (A, D) y se adhirieron sobre fibroblastos placentarios humanos. Tras 18 (B) y 21 (E) días de derivación, las colonias de células tenían el aspecto típico de las CMEh, que se mantuvo tras al menos 90 días de cultivo celular (C, F). Las figuras 2A-J muestran varias características de VAL-1 y VAL-2, incluyendo los cariotipos (A, F), Ia inmunotinción para determinar el antígeno embrionario específico de fase 4 (B, G), TRA-1-60 (C, H) y TRA-1-81 (D, I), y el ensayo de Ia fosfatasa alcalina (E, J). Ambas líneas celulares tenían cariotipos normales y fueron positivas para todos los marcadores probados de no diferenciación (es decir, inmaduros).Figures 1A-F are a series of photographs that show the morphological characteristics of the derivation of VAL-1 and VAL-2. Two different embryos (A, D) were thawed and adhered to human placental fibroblasts. After 18 (B) and 21 (E) days of shunt, the cell colonies had the typical appearance of the CMEh, which was maintained after at least 90 days of cell culture (C, F). Figures 2A-J show several characteristics of VAL-1 and VAL-2, including karyotypes (A, F), immunostaining for determining the specific embryonic antigen of phase 4 (B, G), TRA-1-60 (C , H) and TRA-1-81 (D, I), and the alkaline phosphatase assay (E, J). Both cell lines had normal karyotypes and were positive for all tested markers of non-differentiation (i.e., immature).
La figura 3 muestra Ia actividad telomerasa de las líneas de CMEh VAL-Figure 3 shows the telomerase activity of the lines of CMEh VAL-
1 y VAL-2. El control negativo se obtuvo mediante inactivación térmica en ambas líneas. La banda de 36 pb corresponde al control interno de Ia reacción en cadena de Ia polimerasa y Ia banda de 50 pb corresponde a Ia actividad telomerasa, que aumenta en bandas de 6 pb en las células inmortales.1 and VAL-2. The negative control was obtained by thermal inactivation in both lines. The 36 bp band corresponds to the internal control of the polymerase chain reaction and the 50 bp band corresponds to the telomerase activity, which increases in 6 bp bands in the immortal cells.
La figura 4 es un listado de GeneScanMR que demuestra los perfiles de huella genética de muestras de donantes de VAL-1 (tercera fila), VAL-2 (cuarta fila), materna (donante del óvulos) (primera fila) y paterna (donante de espermatozoides) (segunda fila).Figure 4 is a list of GeneScan MR showing the genetic fingerprint profiles of donor samples from VAL-1 (third row), VAL-2 (fourth row), maternal (egg donor) (first row) and paternal ( sperm donor) (second row).
La figura 5 es un listado de GeneScanMR que demuestra los perfiles de huella genética de VAL-1 y VAL-2. La figura muestra un análisis detallado de los loci D351358 y VWA de Ia tercera y cuarta filas de Ia figura 4.Figure 5 is a list of GeneScan MR showing the genetic fingerprint profiles of VAL-1 and VAL-2. The figure shows a detailed analysis of loci D351358 and VWA of the third and fourth rows of Figure 4.
La figura 6 es una comparación de perfil genómico de VAL-1 y VAL-2 que demuestra que las líneas no son genéticamente idénticas entre sí. Los puntos de datos rodeados con círculos representan niveles de loci genómicos variantes o dispares entre las líneas.Figure 6 is a genomic profile comparison of VAL-1 and VAL-2 that shows that the lines are not genetically identical to each other. Data points surrounded by circles represent varying or different genomic loci levels between the lines.
Las figuras 7A-D son fotografías de miocardiocitos diferenciados derivados espontáneamente de VAL-1. Las figuras 8A-C son fotografías de progenie inmunoteñida diferenciada espontáneamente de VAL-1 y VAL-2. La figura 8B muestra Ia expresión de tubulina B en células similares a neuronas. La figura 8C muestra Ia expresión de actina en células similares a las del músculo.Figures 7A-D are photographs of differentiated cardiomyocytes spontaneously derived from VAL-1. Figures 8A-C are photographs of spontaneously differentiated immunotened progeny of VAL-1 and VAL-2. Figure 8B shows the expression of tubulin B in neuron-like cells. Figure 8C shows the expression of actin in cells similar to those of muscle.
Las figuras 9A-D son fotografías de teratomas formados tras Ia inyección intratesticular de VAL-1 y VAL-2 en ratones SCID.Figures 9A-D are photographs of teratomas formed after the intrathestic injection of VAL-1 and VAL-2 in SCID mice.
Las figuras 10A-L son fotografías que muestran Ia histología de un teratoma derivado de VAL-1. Debe entenderse que los dibujos no son necesarios para posibilitar Ia invención.Figures 10A-L are photographs showing the histology of a teratoma derived from VAL-1. It should be understood that the drawings are not necessary to enable the invention.
Descripción detallada de la invención La invención parte de Ia premisa, en parte, de Ia derivación, Ia caracterización y el uso de líneas de CME procedentes de embriones humanos. Estas líneas se derivan de embriones humanos que se han crioconservado durante varios años. La invención proporciona una caracterización molecular y funcional de estas líneas y de ese modo, abarca líneas de células madre embrionarias que tienen características similares. La invención abarca además métodos de manipulación de tales líneas, incluyendo el cultivo (que incluye Ia propagación), el almacenamiento a largo plazo (que incluye Ia crioconservación), las pruebas (por ejemplo, en un ensayo de detección), y Ia diferenciación de tales líneas. Las líneas de CMEh se generaron a partir de embriones con origen idéntico (es decir, obtenidos a partir de los mismos progenitores (es decir, fuentes de óvulos y espermatozoides)). En consecuencia, los embriones y las líneas se denominan en el presente documento líneas y embriones "hermanos". Además, sin embargo, las líneas también son inmunológicamente idénticas entre sí, tal como se describe en el presente documento. Como resultado, Ia invención prevé el uso combinado de tales líneas en regímenes de tratamiento dada su histocompatibilidad.DETAILED DESCRIPTION OF THE INVENTION The invention starts from the premise, in part, of the derivation, characterization and use of CME lines from human embryos. These lines are derived from human embryos that have been cryopreserved for several years. The invention provides a molecular and functional characterization of these lines and thereby encompasses embryonic stem cell lines that have similar characteristics. The invention also encompasses methods of handling such lines, including cultivation (which includes propagation), long-term storage (which includes cryopreservation), tests (for example, in a detection test), and the differentiation of such lines. The CMEh lines were generated from embryos with identical origin (i.e., obtained from the same progenitors (i.e., sources of ovules and sperm)). Consequently, embryos and lines are referred to herein as "brothers" lines and embryos. In addition, however, the lines are also immunologically identical to each other, as described herein. As a result, the invention provides for the combined use of such lines in treatment regimens given their histocompatibility.
La invención proporciona líneas de CME de origen humano. Tal como se usa en el presente documento, una línea de CME es una línea celular derivada in vitro a partir de un embrión y que tiene propiedades similares a las de las células madre. Las propiedades de las células madre de las líneas de CMEh de Ia invención, incluyen una o más de las propiedades genotípicas, fenotípicas y funcionales descritas en el presente documento. Preferiblemente, Ia línea mantiene tales propiedades a largo plazo y a través de varios pases, cultivos y ciclos de congelación y descongelación. Por ejemplo, VAL-1 se ha sometido a pases al menos 120 veces y VAL-2 se ha sometido a pases al menos 85 veces manteniendo sus propiedades fenotípicas y funcionales. Las propiedades genotípicas pueden ser globales, como el cariotipo, o más detalladas, tal como los perfiles genómlcos. Las líneas facilitadas en el presente documento contienen preferiblemente un cariotipo normal. Las realizaciones específicas de estas líneas tienen un cariotipo 46, XX. El análisis del cariotipo se conoce en Ia técnica y es habitual para los expertos en Ia técnica. Las propiedades fenotípicas incluyen propiedades morfológicas tales como una forma de célula pequeña y redonda con una razón alta de núcleo con respecto a citoplasma y nucléolos prominentes. Las propiedades fenotípicas también incluyen Ia expresión de marcadores intracelulares o de Ia superficie celular. Estas propiedades incluyen Ia expresión de marcadores inmaduros o de no diferenciación, tales como el antígeno embrionario específico de fase 4 (SSEA 4), los antígenos asociados a queratín sulfato, el antígeno de rechazo tumoral (TRA)-I -60 y TRA-1-81, el factor de transcripción celular Oct-4, Rex-1 , Cripto, Thy-1 y/o Nanog. Las líneas de CMEh también pueden caracterizarse según Ia no expresión de determinados marcadores tales como los marcadores de diferenciación. Para este fin, las líneas facilitadas en el presente documento están caracterizadas como que no expresan los marcadores de diferenciación Matni , Amylase y Dbh. Otras propiedades fenotípicas incluyen Ia presencia de fosfatasa alcalina y telomerasa. Los marcadores fenotípicos pueden someterse a ensayo de varias formas. Por ejemplo, pueden someterse a ensayo utilizando agentes de unión específicos de marcador, tales como los anticuerpos específicos de marcador o fragmentos de los mismos. Por ejemplo, SSEA-4 puede detectarse mediante inmunotinción utilizando anticuerpos monoclonales tales como MC-813-70 (Salter y Knowles, 1979) que está comercialmente disponible de Chemicon (Temecula, CA). La expresión de TRA-1-60 y TRA-1-81 puede detectarse utilizando anticuerpos monoclonales disponibles también de fuentes comerciales tales como Chemicon (Temecula, CA). Nanog también puede detectarse utilizando anticuerpos tales como AF1997 disponible de R&D Systems (Minneapolis, MN). Oct-4 también puede detectarse utilizando anticuerpos tales como AF1759 de R&D Systems (Minneapolis, MN), o sc-8628 o sc-9081 disponibles de Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Los marcadores fenotípicos también pueden someterse a ensayo utilizando Ia transcriptasa inversa - reacción en cadena de Ia polimerasa (RT- PCR). Marcadores tales como Oct-4, Rex-1 , Cripta, Thy-1 , Nanog, Matni , Amylase y Dbh pueden detectarse utilizando RT-PCR. Los cebadores para su uso en Ia amplificación mediante Ia RT-PCR de estos diversos marcadores se conocen en Ia técnica. (Véase, por ejemplo, Brimble et al. Stem CeIIs and Development, 13:585-596, 2004; Noaksson et al. Stem CeIIs, 23:1460-1467, 2005.) Alternativamente, tales cebadores pueden diseñarse por un experto habitual en Ia técnica utilizando experimentación de rutina. Por ejemplo, tal como se muestra en los ejemplo, los cebadores pueden diseñarse utilizando software basado en Ia red disponible gratuitamente (Primer3, Genefisher).The invention provides CME lines of human origin. As used herein, a CME line is a cell line derived in vitro from an embryo and having properties similar to those of stem cells. The properties of the stem cells of the CMEh lines of the invention include one or more of the genotypic, phenotypic and functional properties described herein. Preferably, the line maintains such properties in the long term and through several passes, cultures and freezing and thawing cycles. For example, VAL-1 has undergone passes at least 120 times and VAL-2 has undergone passes at least 85 times while maintaining its phenotypic and functional properties. The properties Genotypic can be global, such as the karyotype, or more detailed, such as genomic profiles. The lines provided herein preferably contain a normal karyotype. The specific embodiments of these lines have a 46, XX karyotype. The karyotype analysis is known in the art and is common for those skilled in the art. Phenotypic properties include morphological properties such as a small round cell shape with a high nucleus ratio with respect to cytoplasm and prominent nucleoli. Phenotypic properties also include the expression of intracellular markers or cell surface. These properties include the expression of immature or non-differentiation markers, such as the phase 4 specific embryonic antigen (SSEA 4), the keratin sulfate associated antigens, the tumor rejection antigen (TRA) -I-60 and TRA-1 -81, Oct-4, Rex-1, Crypto, Thy-1 and / or Nanog cell transcription factor. The CMEh lines can also be characterized according to the non-expression of certain markers such as differentiation markers. For this purpose, the lines provided in this document are characterized as not expressing the differentiation markers Matni, Amylase and Dbh. Other phenotypic properties include the presence of alkaline phosphatase and telomerase. Phenotypic markers can be tested in several ways. For example, they can be tested using specific marker binding agents, such as specific marker antibodies or fragments thereof. For example, SSEA-4 can be detected by immunostaining using monoclonal antibodies such as MC-813-70 (Salter and Knowles, 1979) that is commercially available from Chemicon (Temecula, CA). TRA-1-60 and TRA-1-81 expression can be detected using monoclonal antibodies also available from commercial sources such as Chemicon (Temecula, CA). Nanog can also be detected using antibodies such as AF1997 available from R&D Systems (Minneapolis, MN). Oct-4 can also be detected using antibodies such as AF1759 from R&D Systems (Minneapolis, MN), or sc-8628 or sc-9081 available from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phenotypic markers can also be tested using reverse transcriptase - polymerase chain reaction (RT-PCR). Markers such as Oct-4, Rex-1, Crypt, Thy-1, Nanog, Matni, Amylase and Dbh can be detected using RT-PCR. The primers for use in the amplification by means of the RT-PCR of these various markers are known in the art. (See, for example, Brimble et al. Stem CeIIs and Development, 13: 585-596, 2004; Noaksson et al. Stem CeIIs, 23: 1460-1467, 2005.) Alternatively, such primers can be designed by a regular expert in The technique using routine experimentation. For example, as shown in the examples, the primers can be designed using software based on the network available for free (Primer3, Genefisher).
Todavía pueden detectarse otros marcadores fenotípicos utilizando ensayos enzimáticos. Tales marcadores incluyen fosfatasa alcalina y telomerasa. La fosfatasa alcalina puede detectarse en un ensayo convencional utilizando reactivos disponibles de fuentes comerciales tales como Chemicon (Temecula, CA) o Vector Laboratories (Burlingame, CA; Vector Blue/Red Alkaline Phosphatase Substrate Kit). La actividad telomerasa puede detectarse utilizando kits de detección de telomerasa disponibles comercialmente, tales como los disponibles de Chemicon (TRAPEZE Telomerase Detection Kit, Temecula, CA), opcionalmente junto con tinción en gel de ADN, tal como, pero sin limitarse a, tinción en gel SYBR (Molecular Probes, Eugene, OR).Other phenotypic markers can still be detected using enzymatic assays. Such markers include alkaline phosphatase and telomerase. Alkaline phosphatase can be detected in a conventional assay using reagents available from commercial sources such as Chemicon (Temecula, CA) or Vector Laboratories (Burlingame, CA; Vector Blue / Red Alkaline Phosphatase Substrate Kit). Telomerase activity can be detected using commercially available telomerase detection kits, such as those available from Chemicon (TRAPEZE Telomerase Detection Kit, Temecula, CA), optionally together with DNA gel staining, such as, but not limited to, staining in SYBR gel (Molecular Probes, Eugene, OR).
Las propiedades funcionales de las CMEh incluyen Ia capacidad de formar colonias compactas in vitro, Ia capacidad de cultivarse a largo plazo in vitro y, Io que es más importante, Ia capacidad de mantener las características de las células madre a largo plazo, tal como, pero sin limitarse a, Ia pluripotencia, tal como se describe en el presente documento. Estas propiedades pueden someterse a ensayo visualmente, o mediante cultivo y/o diferenciación in vitro o in vivo.The functional properties of the CMEh include the ability to form compact colonies in vitro, the ability to grow in the long term in vitro and, more importantly, the ability to maintain the characteristics of the stem cells in the long term, such as, but not limited to, pluripotence, as described herein. These properties can be tested visually, or by culture and / or differentiation in vitro or in vivo.
Las CMEh son al menos pluripotentes y, en algunos casos, pueden ser totipotentes. La pluripotencia se refiere a Ia capacidad de estas células para generar Ia mayor parte, si no Ia totalidad, de los tejidos en un organismo. La totipotencia se refiere a Ia capacidad de estas células para generar un organismo entero. Las CMEh de Ia invención pueden diferenciarse en linajes de mesodermo, endodermo y ectodermo. En consecuencia, las CMEh pueden diferenciarse en al menos un linaje de mesodermo tal como hueso, cartílago, músculo liso, músculo cardiaco, músculo esquelético, riñon, músculo estriado y células hematopoyéticas; al menos un linaje de endodermo tal como hígado, páncreas, glándula tiroides, intestino primitivo y epitelio respiratorio; y al menos un linaje de ectodermo tal como piel, células de pigmento tales como melanocitos, neuronas, células de Ia glía, folículos pilosos y brotes dentales. Las CMEh también pueden diferenciarse en células germinativas. Las líneas de CMEh pueden existir en cultivo durante periodos de tiempo prolongados (por ejemplo, hasta un año o más, y potencialmente de manera indefinida) sin diferenciarse completamente y sin agotamiento, y manteniendo su fenotipo original, por ejemplo tal como se describe en el presente documento. En consecuencia, Ia invención proporciona líneas de CMEh que tienen una o más de las características anteriores. Preferiblemente, las líneas de CMEh poseen todas las características anteriores, sin embargo, Ia invención no se limita a ellas.The CMEh are at least pluripotent and, in some cases, they can be totipotent. Pluripotence refers to the ability of these cells to generate most, if not all, of the tissues in an organism. Totipotence refers to the ability of these cells to generate a whole organism The CMEh of the invention can be differentiated into mesoderm, endoderm and ectoderm lineages. Consequently, CMEh can differentiate into at least one mesoderm lineage such as bone, cartilage, smooth muscle, heart muscle, skeletal muscle, kidney, striated muscle and hematopoietic cells; at least one endoderm lineage such as liver, pancreas, thyroid gland, primitive intestine and respiratory epithelium; and at least one ectoderm lineage such as skin, pigment cells such as melanocytes, neurons, glia cells, hair follicles and dental buds. CMEh can also differentiate into germ cells. The CMEh lines may exist in culture for prolonged periods of time (for example, up to a year or more, and potentially indefinitely) without fully differentiating and without depletion, and maintaining their original phenotype, for example as described in the present document Consequently, the invention provides CMEh lines that have one or more of the above characteristics. Preferably, the CMEh lines have all the above characteristics, however, the invention is not limited to them.
Las líneas de CMEh facilitadas por Ia invención incluyen las designadas VAL-1 y VAL-2. VAL-1 y VAL-2 son líneas celulares hermanas en el sentido de que se derivan de embriones hermanos (es decir, embriones que tienen los mismos "padres"). Los perfiles de huella genética de marcador para los padres y las líneas celulares se muestran en Ia figura 4. Se analizaron nueve marcadores polimórficos diferentes que tienen transmisión hereditaria independiente (es decir, D3S1358, vWA, FGA, D8D1179, D21S11 , D18S51 , D5S818, D13S317, D7S820). Los datos demuestran, con un 99% de probabilidad, que las líneas celulares VAL-1 y VAL-2 se derivaban de Ia fuente de muestras maternas y paternas mostradas, y en consecuencia que VAL-1 y VAL-2 son líneas celulares hermanas. VAL-1 y VAL-2 también son inmunológicamente idénticos. Las líneas comparten marcadores de HLA idénticos A2, A23, B40, B44, CW4, CW5, DR7, DR15, DQ2 y DQ6. Sin embargo, las líneas son genéticamente diferentes, tal como se demuestra por Ia comparación de perfil genómico de Ia figura 6. Por tanto, las células pueden utilizarse para determinar Ia importancia de tales diferencias genéticas sobre Ia funcionalidad de las líneas, tal como, pero sin limitarse a, Ia capacidad de diferenciación. En algunos casos, Ia invención proporciona las líneas de CMEh en una forma aislada. Tal como se usa en el presente documento, aislado significa que las células están separadas físicamente de su entorno natural, tal como un blastocisto, una MCI (masa celular interna) y componentes de los mismos. Las líneas se facilitan generalmente como una población clonal. Las líneas de CMEh de Ia invención pueden caracterizarse adicionalmente mediante el método de su derivación. VAL-1 y VAL-2 se derivaron de embriones crioconservados a largo plazo (es decir, 5 años o más) en Ia fase de blastocisto. Embriones humanos en el día 2, crioconservados y donados para investigación, se congelaron y después se trataron para eliminar Ia zona pelúcida utilizando, por ejemplo, solución acida de Tyrode. Los embriones se lavaron en medio (por ejemplo, 80% de DMEM, 20% de reposición de suero deficiente libre de suero (GibcoBRL), opcionalmente complementado con aminoácidos no esenciales 0,1 mM, beta-mercaptoetanol 0,1 mM y L-glutamina 1 mM), que contenía opcionalmente factor de crecimiento de fibroblastos básico humano 12 ng/mL (Invitrogen). Los embriones se transfirieron entonces sobre células alimentadoras humanas. Células alimentadoras humanas adecuadas incluyen, pero no se limitan a, los fibroblastos placentarios humanos. Estas células alimentadoras se inactivaron mitóticamente. La activación mitótica puede llevarse a cabo utilizando, por ejemplo, irradiación o productos químicos (por ejemplo, mitomicina C). Los cultivos se mantuvieron durante 2 - 3 semanas, momento en el que las colonias iniciales se alteraron mecánicamente y se les permitió volver a unirse a las células alimentadoras. Tras aproximadamente una semana, las colonias se alteraron mecánicamente de nuevo y después se transfirieron a células alimentadoras frescas. Las CMEh se identificaron morfológicamente como células redondas con nucléolos prominentes. Las colonias individuales se aislaron y se volvieron a cultivar con el fin de lograr una población clonal, tras Io que las líneas se pudieron someter al menos a 50, 75, 100, o más pases.The CMEh lines provided by the invention include those designated VAL-1 and VAL-2. VAL-1 and VAL-2 are sister cell lines in the sense that they are derived from sister embryos (that is, embryos that have the same "parents"). Marker genetic fingerprint profiles for parents and cell lines are shown in Figure 4. Nine different polymorphic markers that have independent hereditary transmission were analyzed (ie, D3S1358, vWA, FGA, D8D1179, D21S11, D18S51, D5S818, D13S317, D7S820). The data demonstrate, with a 99% probability, that the VAL-1 and VAL-2 cell lines were derived from the source of maternal and paternal samples shown, and consequently that VAL-1 and VAL-2 are sister cell lines. VAL-1 and VAL-2 are also immunologically identical. The lines share identical HLA markers A2, A23, B40, B44, CW4, CW5, DR7, DR15, DQ2 and DQ6. However, the lines are genetically different, such as demonstrated by the comparison of genomic profile of Figure 6. Therefore, cells can be used to determine the importance of such genetic differences on the functionality of the lines, such as, but not limited to, the ability to differentiate. In some cases, the invention provides the CMEh lines in an isolated form. As used herein, "isolated" means that the cells are physically separated from their natural environment, such as a blastocyst, an MCI (internal cell mass) and components thereof. The lines are generally provided as a clonal population. The CMEh lines of the invention can be further characterized by the method of their derivation. VAL-1 and VAL-2 were derived from long-term cryopreserved embryos (that is, 5 years or more) in the blastocyst phase. Human embryos on day 2, cryopreserved and donated for research, were frozen and then treated to remove the zona pellucida using, for example, Tyrode acid solution. The embryos were washed in medium (for example, 80% DMEM, 20% serum-free deficient serum replacement (GibcoBRL), optionally supplemented with 0.1 mM non-essential amino acids, 0.1 mM beta-mercaptoethanol and L- 1 mM glutamine), which optionally contained 12 ng / mL basic human fibroblast growth factor (Invitrogen). The embryos were then transferred onto human feeder cells. Suitable human feeder cells include, but are not limited to, human placental fibroblasts. These feeder cells were inactivated mitotically. Mitotic activation can be carried out using, for example, irradiation or chemicals (for example, mitomycin C). The cultures were maintained for 2-3 weeks, at which time the initial colonies were mechanically altered and allowed to rejoin the feeder cells. After about a week, the colonies were mechanically altered again and then transferred to fresh feeder cells. CMEhs were morphologically identified as round cells with prominent nucleoli. The individual colonies were isolated and re-cultivated in order to achieve a clonal population, after Io that the lines could be submitted to at least 50, 75, 100, or more passes.
La invención contempla Ia derivación de las líneas de CMEh facilitadas en el presente documento de otras formas. Como ejemplo, las líneas de CMEh pueden generarse a partir de embriones preparados recientemente o de embriones que se han crioconservado únicamente para un corto plazo (por ejemplo, días, semanas o meses). Como otro ejemplo, el proceso de derivación puede suponer el aislamiento de las células de Ia MCI de un blastocisto sin tratamiento con solución acida de Tyrode.The invention contemplates the derivation of the CMEh lines provided herein in other ways. As an example, the CMEh lines can be generated from newly prepared embryos or from embryos that have been cryopreserved for only a short time (for example, days, weeks or months). As another example, the derivation process can involve the isolation of the MCI cells from a blastocyst without treatment with Tyrode acid solution.
La invención contempla composiciones que comprenden a las CMEh facilitadas en el presente documento. Tales composiciones pueden comprender otros componentes tales como células alimentadoras humanas (por ejemplo, células alimentadoras placentarias humanas), progenie de líneas de células madre, incluyendo progenie diferenciada, factores de diferenciación, matrices extracelulares, vehículos farmacéuticamente aceptables y similares. Estas composiciones pueden incluir cultivos de las CMEh. Tales cultivos pueden incluir suero humano o animal, o pueden estar libres de suero. Por ejemplo, los cultivos pueden comprender reposiciones de suero, tal como se describe en el presente documento.The invention contemplates compositions comprising the CMEhs provided herein. Such compositions may comprise other components such as human feeder cells (eg, human placental feeder cells), progeny of stem cell lines, including differentiated progeny, differentiation factors, extracellular matrices, pharmaceutically acceptable carriers and the like. These compositions may include cultures of the CMEh. Such cultures may include human or animal serum, or they may be serum free. For example, cultures may comprise serum replacements, as described herein.
Por tanto, Ia invención contempla además métodos para cultivar que incluyen Ia propagación de líneas de CMEh no diferenciadas, opcionalmente durante semanas, meses o años. Tal como se trata en los ejemplos, estos métodos y condiciones de cultivo son similares a los métodos y condiciones de derivación previstos en el presente documento. Las líneas pueden cultivarse en presencia de células alimentadoras, preferiblemente células alimentadoras humanas, e incluso más preferiblemente células alimentadoras placentarias humanas, tal como se describe por Genbacev et al. Fértil Steril. 2005, 83:1517- 29. Alternativamente, las células pueden cultivarse en presencia de otros tipos de células alimentadoras humanas incluyendo, pero sin limitarse a, células de músculo, piel, epitelio de Ia trompa de Falopio, endometrio glandular, endometrio estromal, estroma de Ia médula y prepucio, fetales y adultos. Un medio de cultivo adecuado puede comprender DMEM con 20% de reposición de suero deficiente y complementación opcional con aminoácidos no esenciales, beta-mercaptoetanol y L-glutamina. Otros medios base incluyen G2.2, S2 (Scandanavian-2), y similares. Todavía en otras realizaciones, las líneas descritas pueden cultivarse en condiciones libres de células alimentadoras, opcionalmente en presencia de uno o más factores de crecimiento que sustituyen a las células alimentadoras. Tales factores incluyen el factor de crecimiento de fibroblastos (FGF) en cualquiera de sus diversas formas u homólogos del mismo, incluyendo el FGF ácido (o FGFa o FGF1 ) y el FGF básico (o FGFb o FGF2). En algunas realizaciones importantes, el FGF es FGFb. La cantidad de FGF puede variar y un experto habitual en Ia técnica puede determinar Ia cantidad requerida para Ia derivación y/o el cultivo en un estado no diferenciado. Intervalos adecuados incluyen 1 - 1000 ng/mL, 1 - 100 ng/mL, 1 - 15 ng/mL, 1 - 10 ng/mL y 1 - 5 ng/mL. El FGF humano se prefiere en algunas realizaciones. Las CMEh pueden propagarse en cultivo indefinidamente con pases regulares, opcionalmente sobre células alimentadoras frescas.Therefore, the invention also contemplates cultivation methods that include the propagation of undifferentiated CMEh lines, optionally for weeks, months or years. As discussed in the examples, these culture methods and conditions are similar to the derivation methods and conditions provided herein. The lines can be cultured in the presence of feeder cells, preferably human feeder cells, and even more preferably human placental feeder cells, as described by Genbacev et al. Fertile Steril. 2005, 83: 1517-29. Alternatively, cells can be cultured in the presence of other types of human feeder cells including, but not limited to, muscle cells, skin, fallopian tube epithelium, glandular endometrium, stromal endometrium, stroma of the medulla and foreskin, fetals and adults. A suitable culture medium may comprise DMEM with 20% poor serum replacement and optional supplementation with non-amino acids. essentials, beta-mercaptoethanol and L-glutamine. Other base media include G2.2, S2 (Scandanavian-2), and the like. In still other embodiments, the described lines can be grown under conditions free of feeder cells, optionally in the presence of one or more growth factors that replace the feeder cells. Such factors include fibroblast growth factor (FGF) in any of its various forms or homologs thereof, including acidic FGF (or FGFa or FGF1) and basic FGF (or FGFb or FGF2). In some important embodiments, the FGF is FGFb. The amount of FGF can vary and a person skilled in the art can determine the amount required for the derivation and / or culture in an undifferentiated state. Suitable ranges include 1 - 1000 ng / mL, 1 - 100 ng / mL, 1 - 15 ng / mL, 1 - 10 ng / mL and 1 - 5 ng / mL. Human FGF is preferred in some embodiments. The CMEh can be propagated in culture indefinitely with regular passes, optionally on fresh feeder cells.
Los métodos de derivación y propagación facilitados en el presente documento en algunos casos, utilizan células alimentadoras tales como células alimentadoras humanas, y no requieren el uso de suero animal. Por tanto, Ia probabilidad de contaminación de especies cruzada utilizando estos métodos es de baja a inexistente. En consecuencia, las líneas de CMEh y las composiciones de las mismas pueden caracterizarse adicionalmente por Ia ausencia de patógenos animales y células animales o subproductos celulares.The derivation and propagation methods provided herein in some cases use feeder cells such as human feeder cells, and do not require the use of animal serum. Therefore, the probability of cross-species contamination using these methods is low to non-existent. Consequently, the CMEh lines and their compositions can be further characterized by the absence of animal pathogens and animal cells or cellular by-products.
La invención proporciona el uso de líneas de CMEh generadas en el plazo de meses o años de su derivación. Por tanto, las líneas de CMEh pueden almacenarse indefinidamente tal como mediante crioconservación. Los métodos para crioconservar CME se conocen en Ia técnica. La congelación de las células puede llevarse a cabo utilizando métodos que incluyen, pero sin limitarse a, métodos de congelación lenta convencional utilizando dimetilsulfóxido (DMSO, preferiblemente al 10%) como agente de crioconservación (tal como se describe por Bongso et al. Hum. Reprod. 9(11): 2110-2117, 1994), métodos de vitrificación (tal como se describe por Reubinoff et al. Hum. Reprod. 16(10): 2187-2194, 2001), así como otros métodos, tales como los descritos por Ji et al. Biotechnol Bioeng (2004), 5:299-312, y Richards et al. Stem CeIIs (2004); 22: 779-789. Sin embargo, debe entenderse que en algunas realizaciones las líneas de CMEh pueden utilizarse antes de Ia crioconservación, y directamente a partir del cultivo. La invención no está limitada de esta manera.The invention provides the use of CMEh lines generated within months or years of their derivation. Therefore, the CMEh lines can be stored indefinitely such as by cryopreservation. The methods to cryopreserve CME are known in the art. Cell freezing can be carried out using methods that include, but are not limited to, conventional slow freezing methods using dimethylsulfoxide (DMSO, preferably 10%) as a cryopreservation agent (as described by Bongso et al. Hum. Reprod. 9 (11): 2110-2117, 1994), vitrification methods (as described by Reubinoff et al. Hum. Reprod. 16 (10): 2187-2194, 2001), as well as other methods, such as described by Ji et al. Biotechnol Bioeng (2004), 5: 299-312, and Richards et al. Stem CeIIs (2004); 22: 779-789. However, it should be understood that in some embodiments the CMEh lines can be used before cryopreservation, and directly from the culture. The invention is not limited in this way.
Las líneas de CMEh pueden utilizarse tanto con fines terapéuticos como de investigación. Las líneas pueden diferenciarse en uno o más linajes. Las propias CMEh y/o su progenie pueden utilizarse terapéuticamente. Alternativamente, las CMEh y/o su progenie pueden utilizarse in vitro para varios fines, incluyendo Ia detección e identificación de factores de autorrenovación y factores de diferenciación, y para someter a prueba diversos factores, incluyendo supuestos compuestos candidatos terapéuticos.The CMEh lines can be used for both therapeutic and research purposes. The lines can be differentiated into one or more lineages. The CMEh itself and / or its progeny can be used therapeutically. Alternatively, the CMEh and / or their progeny can be used in vitro for various purposes, including the detection and identification of self-renewal factors and differentiation factors, and to test various factors, including supposed therapeutic candidate compounds.
La invención contempla métodos para diferenciar las líneas de CMEh en uno o más linajes particulares incluyendo, pero sin limitarse a, células endoteliales, neuronas, células hematopoyéticas, cardiomiocitos, células del músculo esquelético, hepatocitos, células productoras de insulina, células progenitoras de Ia glía, osteoblastos, gametos y células renales. Las líneas de CMEh pueden utilizarse para regenerar un(o) linaje(s) celulares) específico(s), tejido u órgano. La invención engloba además Ia progenie diferenciada resultante que incluye, hueso, cartílago, músculo liso, músculo cardiaco, músculo esquelético, riñon, músculo estriado y células hematopoyéticas (linajes mesodérmicos), hígado, páncreas, glándula tiroides, intestino primitivo y epitelio respiratorio (linajes endodérmicos), piel, células de pigmento tales como melanocitos, neuronas, células de Ia glía, folículos pilosos y brotes dentales (linajes ectodérmicos) y los usos de los mismos.The invention contemplates methods for differentiating the CMEh lines in one or more particular lineages including, but not limited to, endothelial cells, neurons, hematopoietic cells, cardiomyocytes, skeletal muscle cells, hepatocytes, insulin producing cells, glial progenitor cells. , osteoblasts, gametes and renal cells. The CMEh lines can be used to regenerate a specific cell lineage (s), tissue or organ. The invention also encompasses the resulting differentiated progeny that includes bone, cartilage, smooth muscle, heart muscle, skeletal muscle, kidney, striated muscle and hematopoietic cells (mesodermal lineages), liver, pancreas, thyroid gland, primitive intestine and respiratory epithelium (lineages endodermal), skin, pigment cells such as melanocytes, neurons, glia cells, hair follicles and dental outbreaks (ectodermal lineages) and their uses.
La diferenciación puede producirse in vitro o in vivo, dependiendo de Ia aplicación. Se han descrito métodos para diferenciar las CMEh ¡n vitro. Las CMEh se diferencian mediante su exposición a condiciones de diferenciación durante un tiempo suficiente para producir células diferenciadas. Tal como se usa en el presente documento, condiciones de diferenciación son condiciones que inducen a las CMEh a que se diferencien en uno o más linajes. Estas condiciones pueden variar según el linaje deseado. En general, estas condíciones pueden comprender Ia ausencia de células alimentadoras utilizadas para mantener a las CMEh en una forma no diferenciada, los cambios en Ia densidad de sembrado de las células, y/o Ia introducción de uno o más factores de crecimiento y/o de otras células alimentadoras que estimulan Ia diferenciación en linajes particulares. Los factores de crecimiento que pueden inducir Ia diferenciación de las CMEh en linajes particulares incluyen, pero no se limitan a, ácido retinoico, factor de crecimiento epidérmico (EGF), proteína morfogenética ósea 4 (BMP4), factor de crecimiento de fibroblastos (FGF), hormonas esteroideas (por ejemplo, glucocorticoides, vitamina A, hormona tiroidea, andrógenos, estrógenos y similares), activina-A (mesodermo), factor de crecimiento transformante beta 1 (TGF-/J1) (mesodermo), factor de crecimiento de hepatocitos (HGF), y factor de crecimiento nervioso (NGF).Differentiation can occur in vitro or in vivo, depending on the application. Methods for differentiating in vitro CMEh have been described. The CMEh are differentiated by exposure to differentiation conditions for a sufficient time to produce differentiated cells. As used herein, differentiation conditions are conditions that induce the CMEh to differ in one or more lineages. These conditions may vary according to the desired lineage. In general, you are Conditions may include the absence of feeder cells used to maintain the CMEh in an undifferentiated form, changes in the seeding density of the cells, and / or the introduction of one or more growth factors and / or other feeder cells that stimulate the differentiation in particular lineages. Growth factors that can induce the differentiation of CMEh in particular lineages include, but are not limited to, retinoic acid, epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), fibroblast growth factor (FGF) , steroid hormones (e.g., glucocorticoids, vitamin A, thyroid hormone, androgens, estrogens and the like), activin-A (mesoderm), transforming growth factor beta 1 (TGF- / J1) (mesoderm), hepatocyte growth factor (HGF), and nerve growth factor (NGF).
La diferenciación en músculo cardiaco puede inducirse utilizando ácido retinoico, 5-azacitidina y ácido ascórbico. La diferenciación en linajes hematopoyéticos puede inducirse utilizando células del estroma de Ia médula ósea, tal como se describe en Ia patente de los EE.UU. número 6.613.568 y/o factores hematopoyéticos de acción temprana, tales como el ligando kit, IL-11 , VEGF, el ligando Flk2/Flt3 y similares. La diferenciación en linaje neuronal puede inducirse utilizando EGF y FGFb tal como se describe en Ia solicitud de patente publicada de los EE.UU. número 20050260747.Differentiation in cardiac muscle can be induced using retinoic acid, 5-azacitidine and ascorbic acid. Differentiation in hematopoietic lineages can be induced using bone marrow stromal cells, as described in US Pat. No. 6,613,568 and / or early-acting hematopoietic factors, such as kit ligand, IL-11, VEGF, Flk2 / Flt3 ligand and the like. Differentiation in neuronal lineage can be induced using EGF and FGFb as described in the published US patent application. number 20050260747.
Las líneas de CMEh pueden utilizarse en Ia práctica de transplantes en el tratamiento (incluyendo Ia prevención) de varios estados que afectan a uno o más linajes. Ejemplos de tales estados incluyen, pero no se limitan a, Ia enfermedad de Parkinson (neuronas dopaminérgicas), Ia enfermedad de Alzheimer (precursores neuronales), Ia enfermedad de Huntington (neuronas GABAérgicas), trastornos sanguíneos tales como leucemia, linfoma, mieloma y anemia (células hematopoyéticas), efectos secundarios de Ia radiación, por ejemplo, en pacientes con transplante (precursores hematopoyéticos), infarto de miocardio, tejido cardiaco isquémico o insuficiencia cardiaca (cardiomiocitos parcial o totalmente diferenciados), distrofia muscular (células del músculo esquelético), cirrosis o insuficiencia hepática (hepatocitos), hepatitis crónica (hepatocitos), diabetes incluyendo diabetes tipo I (células productoras de insulina tales como las células de los islotes), lesión cerebral isquémica (neuronas), lesión en Ia médula espinal (células progenitoras de Ia glía y neuronas motoras), esclerosis lateral amiotrófica (ELA) (neuronas motoras), lesión en tejido ortopédico (osteoblastos), lesión renal (células renales), cicatriz corneal (células madre corneales), lesión en el cartílago (condrocitos), lesión ósea (células osteogénicas, incluyendo los osteocitos), osteoartritis (condrocitos), trastornos en Ia mielinización, tales como Ia enfermedad de Pelizaeus-Merzbacher, esclerosis múltiples, adenoleucodistrofias, neuritis y neuropatías (oligodendrocitos) y alopecia. La bibliografía que documenta Ia diferenciación de las células madre embrionarias en estos diversos linajes incluye Bjorklund et al., 2002, PNAS USA 99: 2344-2349 (neuronas dopaminérgicas), West y Daley, 2004, Curr Opin CeII Biol 16: 688-692; Ia patente de los EE.UU. número 6.534.052 B1 ; Kehat y Gepstein, 2003, Heart Fail Rev 8: 229-236; Nir et al., 2003, Cardiovasc Res 58: 313-323; las patentes de los EE.UU. números 6.613.568 y 6.833.269. La invención comprende los transplantes de células diferenciadas y/o células madre embrionarias no diferenciadas o parcialmente diferenciadas.The CMEh lines can be used in the practice of transplants in the treatment (including prevention) of several states that affect one or more lineages. Examples of such conditions include, but are not limited to, Parkinson's disease (dopaminergic neurons), Alzheimer's disease (neuronal precursors), Huntington's disease (GABAergic neurons), blood disorders such as leukemia, lymphoma, myeloma and anemia (hematopoietic cells), side effects of radiation, for example, in patients with transplantation (hematopoietic precursors), myocardial infarction, ischemic heart tissue or heart failure (partially or totally differentiated cardiomyocytes), muscular dystrophy (skeletal muscle cells), cirrhosis or liver failure (hepatocytes), chronic hepatitis (hepatocytes), diabetes including type I diabetes (insulin producing cells such as islet cells), ischemic brain injury (neurons), spinal cord injury (progenitor cells of the glia and motor neurons), amyotrophic lateral sclerosis ( ALS) (motor neurons), orthopedic tissue injury (osteoblasts), renal injury (renal cells), corneal scar (corneal stem cells), cartilage lesion (chondrocytes), bone lesion (osteogenic cells, including osteocytes), osteoarthritis (chondrocytes), disorders in myelination, such as Pelizaeus-Merzbacher disease, multiple sclerosis, adenoleukodystrophies, neuritis and neuropathies (oligodendrocytes) and alopecia. The literature documenting the differentiation of embryonic stem cells in these various lineages includes Bjorklund et al., 2002, PNAS USA 99: 2344-2349 (dopaminergic neurons), West and Daley, 2004, Curr Opin CeII Biol 16: 688-692 ; US Pat. No. 6,534,052 B1; Kehat and Gepstein, 2003, Heart Fail Rev 8: 229-236; Nir et al., 2003, Cardiovasc Res 58: 313-323; U.S. patents Nos. 6,613,568 and 6,833,269. The invention comprises transplants of differentiated cells and / or undifferentiated or partially differentiated embryonic stem cells.
Las CMEh y/o su progenie diferenciada pueden introducirse en un sujeto local o sistémicamente mediante varios métodos y vías. La administración local incluye Ia inyección directa en sitios particulares en el organismo, incluyendo órganos y tejidos ya sean normales o anómalos. Tal administración local puede realizarse mediante inyección directa con aguja. La administración sistémica abarca vías de administración parenteral (por ejemplo, intravenosa, intramuscular, subcutánea, intraperitoneal, intratumoral, intratecal, etc.) y no parenteral.The CMEh and / or its differentiated progeny can be introduced into a subject locally or systemically by various methods and routes. Local administration includes direct injection into particular sites in the body, including normal and abnormal organs and tissues. Such local administration can be done by direct needle injection. Systemic administration encompasses parenteral (eg, intravenous, intramuscular, subcutaneous, intraperitoneal, intratumoral, intrathecal, etc.) and non parenteral administration routes.
Las líneas de CMEh y/o su progenie diferenciada pueden proporcionarse en preparaciones farmacéuticas. Tales preparaciones son adecuadas para Ia administración in vivo y, por tanto, son mínimamente estériles y fisiológicamente aceptables para el receptor. Estas preparaciones pueden comprender generalmente un vehículo farmacéuticamente aceptable. Tal como se usa en el presente documento, un vehículo farmacéuticamente aceptable significa un material no tóxico que no interfiere con Ia eficacia de las células y/o otros agentes administrados. Vehículos farmacéuticamente aceptables incluyen diluyentes, cargas, sales, tampones, estabilizantes, conservantes, solubilizantes y otros materiales que son bien conocidos en Ia técnica. Las sales farmacéuticamente aceptables incluyen, pero no se limitan a, aquellas preparadas a partir de los siguientes ácidos: clorhídrico, bromhídrico, sulfúrico, nítrico, fosfórico, maleico, acético, salicílico, cítrico, fórmico, malónico, succínico y similares. Además, las sales farmacéuticamente aceptables pueden prepararse como sales de metal alcalino o alcalino-térreo, tales como las sales de sodio, potasio o calcio. Las preparaciones farmacéuticas también pueden contener otros agentes terapéuticos. La invención también abarca preparaciones farmacéuticas que se formulan para administración local, tales como implantes. Ejemplos de implantes bioerosionables se describen en Ia solicitud internacional PCT número PCT/US/03307 (número de publicación WO 95/24929).The CMEh lines and / or their differentiated progeny can be provided in pharmaceutical preparations. Such preparations are suitable for administration in vivo and, therefore, are minimally sterile and physiologically acceptable to the recipient. These preparations may generally comprise a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable vehicle means a non-toxic material that does not interfere with the effectiveness of the cells and / or other agents administered. Pharmaceutically acceptable vehicles include diluents, fillers, salts, buffers, stabilizers, preservatives, solubilizers and other materials that are well known in the art. Pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic and the like. In addition, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts. Pharmaceutical preparations may also contain other therapeutic agents. The invention also encompasses pharmaceutical preparations that are formulated for local administration, such as implants. Examples of bioerodible implants are described in the international PCT application number PCT / US / 03307 (publication number WO 95/24929).
La invención contempla además Ia capacidad para transducir células madre embrionarias y/o su progenie diferenciada con ácidos nucleicos particulares, dando lugar así a células madre y progenie modificadas genéticamente. Si están destinadas al transplante, estas células pueden utilizarse, por ejemplo, para generar factores particulares o para complementar mutaciones particulares en el receptor. La transducción de las líneas de CMEh también se enseña en Ia publicación de solicitud de patente de los EE.UU. número 20050079616.The invention also contemplates the ability to transduce embryonic stem cells and / or their differentiated progeny with particular nucleic acids, thus giving rise to genetically modified stem cells and progeny. If they are intended for transplantation, these cells can be used, for example, to generate particular factors or to complement particular mutations in the recipient. The transduction of the CMEh lines is also taught in the US patent application publication. Number 20050079616.
Tal como se usa en el presente documento, "transducción de células madre embrionarias" se refiere al proceso de transferir material genético exógeno en una célula madre embrionaria. Los términos "transducción", "transfección" y "transformación" se utilizan de manera intercambiable en el presente documento, y se refieren al proceso de transferir material genético exógeno a una célula. Tal como se usa en el presente documento, "material genético exógeno" se refiere a ácidos nucleicos u oligonucleótidos, naturales o sintéticos, que se introducen en las células. El material genético exógeno puede ser una copia de éste que está presente de manera natural en las células, o puede no encontrarse de manera natural en las células. Normalmente, es al menos una parte de un gen que se produce de manera natural que se ha colocado bajo el control operativo de un promotor en un constructo de vector. Pueden emplearse varias técnicas para introducir ácidos nucleicos en las células. Tales técnicas incluyen Ia transfección de precipitados de ácido nucleico - CaPO4, Ia transfección de ácidos nucleicos asociados con DEAE, Ia transfección con un retrovirus que incluya al ácido nucleico de interés, Ia transfección mediada por liposomas y similares. Para ciertos usos, se prefiere seleccionar como objetivo el ácido nucleico para células particulares. En tales casos, un vehículo utilizado para administrar un ácido nucleico según Ia invención en una célula (por ejemplo, un retrovirus, u otro virus; un liposoma) puede tener una molécula de selección de diana unida al mismo. Por ejemplo, una molécula tal como un anticuerpo específico para una proteína de Ia membrana superficial en una célula diana o un ligando para un receptor en Ia célula diana, pueden unirse o incorporarse dentro del vehículo de administración del ácido nucleico. Por ejemplo, cuando se emplean liposomas para administrar los ácidos nucleicos de Ia invención, pueden incorporarse proteínas que se unen a una proteína de Ia membrana superficial asociada con endocitosis en Ia formulación del liposoma para seleccionar como diana y/o facilitar Ia captación. Tales proteínas incluyen proteínas o fragmentos de las mismas trópicos para un tipo celular particular, anticuerpos para proteínas que experimentan intemalización, proteínas que confieren localización intracelular y/o que potencian Ia semivida intracelular y similares. También se han utilizado satisfactoriamente sistemas de administración poliméricos para administrar ácidos nucleicos en las células, tal como conocen los expertos en Ia técnica.As used herein, "embryonic stem cell transduction" refers to the process of transferring exogenous genetic material into an embryonic stem cell. The terms "transduction", "transfection" and "transformation" are used interchangeably herein, and refer to the process of transferring exogenous genetic material to a cell. As used herein, "exogenous genetic material" refers to natural or synthetic nucleic acids or oligonucleotides, which are introduced into cells. The exogenous genetic material may be a copy of it that is naturally present in the cells, or may not be found naturally in cells. Normally, it is at least a part of a naturally occurring gene that has been placed under the operational control of a promoter in a vector construct. Several techniques can be used to introduce nucleic acids into cells. Such techniques include the transfection of nucleic acid precipitates - CaPO 4 , the transfection of nucleic acids associated with DEAE, the transfection with a retrovirus that includes the nucleic acid of interest, liposome-mediated transfection and the like. For certain uses, it is preferred to select the target nucleic acid for particular cells. In such cases, a vehicle used to deliver a nucleic acid according to the invention in a cell (for example, a retrovirus, or another virus; a liposome) can have a target selection molecule attached to it. For example, a molecule such as an antibody specific for a surface membrane protein in a target cell or a ligand for a receptor in the target cell, can be attached or incorporated into the nucleic acid delivery vehicle. For example, when liposomes are used to administer the nucleic acids of the invention, proteins can be incorporated that bind to a surface membrane protein associated with endocytosis in the liposome formulation to select as a target and / or facilitate the uptake. Such proteins include proteins or fragments of the same tropics for a particular cell type, antibodies for proteins that undergo intemalization, proteins that confer intracellular localization and / or that enhance intracellular half-life and the like. Polymeric administration systems have also been successfully used to administer nucleic acids in the cells, as is known to those skilled in the art.
Un método para introducir material genético exógeno en las células es mediante el uso de retrovirus deficientes en Ia replicación. Los retrovirus deficientes en Ia replicación pueden dirigir Ia síntesis de todas las proteínas del virión, pero no pueden formar partículas infecciosas. En consecuencia, estos vectores retrovirales alterados genéticamente tienen una utilidad general para Ia transducción de genes de alta eficacia en células en cultivo, y una utilidad específica para su uso en el método de Ia presente invención. Los retrovirus se han utilizado ampliamente para transferir material genético a las células. Los protocolos convencionales para producir retrovirus deficientes en Ia replicación (incluyendo las etapas de incorporación del material genético exógeno en un plásmido, Ia transfección de una línea celular de empaquetamiento con el plásmido, Ia producción de retrovirus recombinantes mediante Ia línea celular de empaquetamiento, Ia recogida de las partículas virales del medio de cultivo tisular y Ia infección de las células diana con las partículas virales) están previstos en Ia técnica. Un vector de expresión retroviral preferido incluye un elemento promotor exógeno para controlar Ia transcripción del gen exógeno insertado. Tales promotores exógenos incluyen tanto promotores constitutivos como inducibles.A method for introducing exogenous genetic material into cells is through the use of retroviruses deficient in replication. Retroviruses deficient in replication can direct the synthesis of all virion proteins, but cannot form infectious particles. Consequently, these genetically altered retroviral vectors have a general utility for the transduction of genes of high efficiency in cells in culture, and a utility specific for use in the method of the present invention. Retroviruses have been widely used to transfer genetic material to cells. Conventional protocols to produce replication-deficient retroviruses (including the steps of incorporating the exogenous genetic material into a plasmid, the transfection of a packaging cell line with the plasmid, the production of recombinant retroviruses by means of the packaging cell line, the collection of the viral particles of the tissue culture medium and the infection of the target cells with the viral particles) are provided in the technique. A preferred retroviral expression vector includes an exogenous promoter element to control the transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.
La principal ventaja del uso de retrovirus es que el virus inserta eficazmente una única copia del gen que codifica para el agente terapéutico en el genoma de Ia célula huésped, permitiendo así que el material genético exógeno pase a Ia progenie de Ia célula cuando se divide. Además, se ha notificado que las secuencias de promotor génico en Ia región LTR potencian Ia expresión de una secuencia codificante insertada en una variedad de tipos celulares. Las principales desventajas de utilizar un vector de expresión de retrovirus son (1 ) Ia mutagénesis insercional, es decir, Ia inserción del gen terapéutico en una posición no deseada en el genoma de Ia célula diana Io que, por ejemplo, conduce a falta de regulación del crecimiento celular y (2) Ia necesidad de proliferación de Ia célula diana con el fin de que el gen terapéutico portado por el vector se integre en el genoma diana. Pese a estas aparentes limitaciones, Ia administración de una cantidad terapéuticamente eficaz de un agente terapéutico mediante un retrovirus puede ser eficaz si Ia eficiencia de Ia transducción es alta y/o el número de células diana disponibles para Ia transducción es alta.The main advantage of the use of retroviruses is that the virus effectively inserts a single copy of the gene that codes for the therapeutic agent in the genome of the host cell, thus allowing the exogenous genetic material to pass to the progeny of the cell when it is divided. In addition, it has been reported that gene promoter sequences in the LTR region enhance the expression of a coding sequence inserted in a variety of cell types. The main disadvantages of using a retrovirus expression vector are (1) the insertional mutagenesis, that is, the insertion of the therapeutic gene in an undesired position in the genome of the target cell, which, for example, leads to lack of regulation of cell growth and (2) the need for proliferation of the target cell so that the therapeutic gene carried by the vector is integrated into the target genome. Despite these apparent limitations, the administration of a therapeutically effective amount of a therapeutic agent by a retrovirus can be effective if the efficiency of transduction is high and / or the number of target cells available for transduction is high.
Todavía otro candidato viral útil como vector de expresión para Ia transformación de células es el adenovirus, un virus ADN de doble hebra. Al igual que el retrovirus, el genoma del adenovirus se puede adaptar para su uso como un vector de expresión para Ia transducción génica, es decir, eliminando Ia información genética que controla Ia producción del propio virus. Dado que el adenovirus funciona normalmente de una forma extracromosómica, el adenovirus recombinante no tiene el problema teórico de Ia mutagénesis insercional. Por otra parte, Ia transformación con adenovirus de una célula diana puede que no dé como resultado una transducción estable. Sin embargo, se ha notificado más recientemente que ciertas secuencias del adenovirus confieren especificidad de integración intracromosómica para secuencias vehículo y, por tanto, dan como resultado una transducción estable del material genético exógeno. Por tanto, tal como será evidente para un experto en Ia técnica, se dispone de una variedad de vectores adecuados para transferir material genético exógeno en las células. La selección de un vector apropiado para administrar un agente terapéutico para un estado particular que se pueda tratar con terapia de sustitución de genes y Ia optimización de las condiciones para Ia inserción del vector de expresión seleccionado en Ia célula, están dentro del alcance de un experto habitual en Ia técnica, sin necesidad de experimentación excesiva. El promotor tiene característicamente una secuencia de nucleótidos específica necesaria para iniciar Ia transcripción. Opcionalmente, el material genético exógeno incluye además secuencias adicionales (es decir, enhancers (potenciadores)) requeridos para obtener Ia actividad de transcripción génica deseada. Para los fines de esta discusión, un "enhancer" es simplemente cualquier secuencia de ADN no traducida que funcione con Ia secuencia codificante (en cis) para cambiar el nivel de transcripción basal dictado por el promotor. Preferiblemente, el material genético exógeno se introduce en el genoma inmediatamente en el sentido 3' desde el promotor, de manera que el promotor y Ia secuencia codificante se unan operativamente para permitir Ia transcripción de Ia secuencia codificante.Still another viral candidate useful as an expression vector for cell transformation is adenovirus, a double-stranded DNA virus. Like the retrovirus, the adenovirus genome can be adapted for use as an expression vector for gene transduction, that is, by eliminating The genetic information that controls the production of the virus itself. Since the adenovirus normally works in an extrachromosomal way, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis. On the other hand, the transformation with adenovirus of a target cell may not result in a stable transduction. However, it has been reported more recently that certain adenovirus sequences confer intrachromosomal integration specificity for vehicle sequences and, therefore, result in a stable transduction of exogenous genetic material. Therefore, as will be apparent to one skilled in the art, a variety of vectors suitable for transferring exogenous genetic material into the cells are available. The selection of an appropriate vector to administer a therapeutic agent for a particular state that can be treated with gene replacement therapy and the optimization of the conditions for the insertion of the selected expression vector in the cell, are within the scope of an expert. usual in the technique, without the need for excessive experimentation. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material also includes additional sequences (ie enhancers (enhancers)) required to obtain the desired gene transcription activity. For the purposes of this discussion, an "enhancer" is simply any non-translated DNA sequence that works with the coding sequence (in cis) to change the level of basal transcription dictated by the promoter. Preferably, the exogenous genetic material is introduced into the genome immediately in the 3 'direction from the promoter, so that the promoter and the coding sequence are operatively linked to allow transcription of the coding sequence.
Los promotores constitutivos que se producen en Ia naturaleza controlan Ia expresión de las funciones esenciales de Ia célula. Como resultado, un gen bajo el control de un promotor constitutivo se expresa en todas las condiciones del crecimiento celular. Promotores constitutivos ejemplo incluyen los promotores de los genes siguientes que codifican para ciertas funciones constitutivas o "housekeeping": hipoxantina fosforribosil tranfθrasa (HPRT), dihidrofolato reductasa (DHFR) (Scharfmann et al., Proc. Nati. Acad. ScL USA 88: 4626-4630 (1991)), adenosina desaminasa, fosfoglicerol cinasa (PGK), piruvato cinasa, fosfoglicerol mutasa, el promotor de Ia actina (Lai et al., Proc. Nati. Acad. Sci. USA 86: 10006-10010 (1989)), y otros promotores constitutivos conocidos por el experto en Ia técnica. Además, muchos promotores virales funcionan de manera constitutiva en células eucariotas. Estos incluyen: los promotores temprano y tardío de SV40; las repeticiones terminales largas (LTRS) del virus de Ia leucemia de Moloney y otros retrovirus; y el promotor de Ia timidina cinasa del virus Herpes simplex, entre muchos otros. En consecuencia, cualquiera de los promotores constitutivos nombrados anteriormente puede utilizarse para controlar Ia transcripción de un inserto de gen heterólogo.The constitutive promoters that occur in nature control the expression of the essential functions of the cell. As a result, a gene under the control of a constitutive promoter is expressed in all cell growth conditions. Example constitutive promoters include promoters of the following genes that code for certain functions constitutive or "housekeeping": hypoxanthine phosphoribosyl tranfθrase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., Proc. Nati. Acad. ScL USA 88: 4626-4630 (1991)), adenosine deaminase, phosphoglycerol kinase (PGK) , pyruvate kinase, phosphoglycerol mutase, the actin promoter (Lai et al., Proc. Nati. Acad. Sci. USA 86: 10006-10010 (1989)), and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: SV40 early and late promoters; long terminal repetitions (LTRS) of Moloney leukemia virus and other retroviruses; and the thymidine kinase promoter of the Herpes simplex virus, among many others. Consequently, any of the constitutive promoters named above can be used to control the transcription of a heterologous gene insert.
Los genes que están bajo el control de promotores inducibles se expresan sólo o en un mayor grado, en presencia de un agente de inducción, (por ejemplo, Ia transcripción bajo el control del promotor de Ia metalotioneína se aumenta enormemente en presencia de ciertos iones metálicos). Los promotores inducibles incluyen elementos de respuesta (ER) que estimulan Ia transcripción cuando se unen sus factores de inducción. Por ejemplo, hay ER para factores séricos, hormonas esteroideas, ácido retinoico y AMP cíclico. Los promotores que contienen un ER particular pueden escogerse con el fin de obtener una respuesta inducible y, en algunos casos, el propio ER puede unirse a un promotor diferente, confiriendo así capacidad de inducción al gen recombinante. Por tanto, mediante Ia selección del promotor apropiado (constitutivo frente a inducible; fuerte frente a débil), es posible controlar tanto Ia existencia como el nivel de expresión de un agente terapéutico en Ia célula genéticamente modificada. La selección y Ia optimización de estos factores para Ia administración de una dosis terapéuticamente eficaz de un agente terapéutico particular se considera que está dentro del alcance de un experto habitual en Ia técnica sin experimentación excesiva, teniendo en cuenta los factores descritos anteriormente y el perfil clínico del paciente.The genes that are under the control of inducible promoters are expressed only or to a greater degree, in the presence of an induction agent, (for example, the transcription under the control of the metallothionein promoter is greatly increased in the presence of certain metal ions ). Inducible promoters include response elements (ER) that stimulate transcription when their induction factors are joined. For example, there are ER for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular ER can be chosen in order to obtain an inducible response and, in some cases, the ER itself can bind to a different promoter, thereby conferring induction capacity on the recombinant gene. Therefore, by selecting the appropriate promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and the level of expression of a therapeutic agent in the genetically modified cell. The selection and optimization of these factors for the administration of a therapeutically effective dose of a particular therapeutic agent is considered to be within the scope of a person skilled in the art without excessive experimentation, taking into account the factors described above and the clinical profile. of the patient.
Además de al menos un promotor y al menos un ácido nucleico heterólogo que codifique para el agente terapéutico, el vector de expresión preferiblemente incluye un gel de selección, por ejemplo, un gen de resistencia a neomicina, para facilitar Ia selección de las células que se han transfectado o transducido con el vector de expresión. Alternativamente, las células se transfectan con dos o más vectores de expresión, conteniendo al menos un vector el(los) gen(es) que codifica(n) para el (los) agente(es) terapéutico(s), conteniendo el otro vector un gen de selección. La selección de un promotor, enhancer, gen de selección y/o secuencia señal adecuados (descritos más abajo) se considera que está dentro del alcance de un experto habitual en Ia técnica sin experimentación excesiva.In addition to at least one promoter and at least one nucleic acid heterologous that codes for the therapeutic agent, the expression vector preferably includes a selection gel, for example, a neomycin resistance gene, to facilitate the selection of cells that have been transfected or transduced with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector containing the gene (s) encoding the therapeutic agent (s), containing the other vector. a selection gene. The selection of a suitable promoter, enhancer, selection gene and / or signal sequence (described below) is considered to be within the scope of a person skilled in the art without excessive experimentation.
La selección y Ia optimización de un vector de expresión particular para expresar un producto génico específico en una célula se lleva a cabo mediante Ia obtención del gen, preferiblemente con una o más regiones control apropiadas (por ejemplo, promotor, secuencia de inserción); Ia preparación de un constructo de vector que comprende el vector en el que se inserta el gen; Ia transfección o transducción de células en cultivo in vitro con el constructo de vector; y Ia determinación de si el producto génico está presente en las células. The selection and optimization of a particular expression vector to express a specific gene product in a cell is carried out by obtaining the gene, preferably with one or more appropriate control regions (for example, promoter, insertion sequence); Ia preparation of a vector construct comprising the vector in which the gene is inserted; The transfection or transduction of cells in in vitro culture with the vector construct; and the determination of whether the gene product is present in the cells.
Tabla 1. Protocolos de terapia génica humana aprobados por RAC: 1990-1994Table 1. Human gene therapy protocols approved by RAC: 1990-1994
Deficiencia Linfocitos autólogos transducidos con el gen ADA 31/7/90 inmunitaria humano combinada grave (SCID) debido a deficiencia de ADA Cáncer avanzado Linfocitos infiltrantes de tumor transducidos con el 31/7/90 gen del factor de necrosis tumoralDeficiency Autologous lymphocytes transduced with the severe combined human immune ADA 31/7/90 gene (SCID) due to ADA deficiency Advanced cancer Tumor infiltrating lymphocytes transduced with the 31/7/90 tumor necrosis factor gene
Cáncer avanzado Inmunización con células cancerígenas autólogas 07/10/91 transducidas con el gen del factor de necrosis tumoralAdvanced cancer Immunization with autologous cancer cells 07/10/91 transduced with the tumor necrosis factor gene
Cáncer avanzado Inmunización con células cancerígenas autólogas 07/10/91 transducidas con el gen de Ia interleucina-2Advanced cancer Immunization with autologous cancer cells 07/10/91 transduced with the interleukin-2 gene
Pacientes Vector retroviral murino que codifica para genes de 07/6/93 asintomáticos VIH-1 [VIH-IT(V)] infectados con VIH-1 SIDA Efectos de una forma transdominante del gen rev 07/6/93 sobre Ia intervención del SIDAPatients Murine retroviral vector that codes for asymptomatic 07/6/93 HIV-1 [HIV-IT (V)] genes infected with HIV-1 AIDS Effects of a transdominant form of the gene rev 07/6/93 on AIDS intervention
Cáncer avanzado Transferencia del gen de resistencia a múltiples 08/6/93 fármacos (RMF) humano Infección por VIH Linfocitos autólogos transducidos con ribozima 10/9/93 catalítico que escinde ARN de VIH-1 (estudio de fase I)Advanced cancer Transfer of multiple resistance gene 08/6/93 human drugs (RMF) HIV infection Autologous lymphocytes transduced with ribozyme 10/9/93 catalytic that cleaves HIV-1 RNA (phase I study)
Melanoma Vacunas tumorales autólogas modificadas mediante 10/9/93 metastático ingeniería genética que producen interleucina-2 Infección por VIH Vector retroviral murino que codifica para genes de 03/12/93 VIH-IT(V) (ensayo de fase I / Il de etiqueta abierta)Melanoma Autologous tumor vaccines modified by 10/9/93 metastatic genetic engineering that produce interleukin-2 HIV infection Murine retroviral vector encoding genes of 03/12/93 HIV-IT (V) (phase I / Il label assay open)
Infección por VIH Transferencia adoptiva de linfocitos T citotóxicos 03/3/94 (gemelos idénticos) singénicos (ensayo piloto de fase I / II) Cáncer de mama Uso de retrovirus modificados para introducir 09/6/94 (quimioprotección secuencias de resistencia a Ia quimioterapia en durante el células hematopoyéticas normales tratamiento) (estudio piloto) Anemia de Fanconi Transferencia génica mediada por retrovirales del 09/6/94 gen del grupo C de complementación de Ia anemia de Fanconi a progenitores hemetopoyéticosHIV infection Adoptive transfer of cytotoxic T lymphocytes 03/3/94 (identical twins) syngeneic (phase I / II pilot trial) Breast cancer Use of modified retroviruses to introduce 9/6/94 (chemoprotection chemotherapy resistance sequences in during normal hematopoietic cells treatment) (pilot study) Fanconi anemia Retroviral mediated gene transfer of 9/6/94 gene group C of complementation of Fanconi anemia to hemetopoietic progenitors
Carcinoma de Gen del factor estimulante de colonias de ORDA/NI próstata metastásico macrófagos-granulocitos humanos autólogos H transducido con Ia vacuna del cáncer de próstata 03/8/94*Gen carcinoma of the ORDA / NI colony stimulating factor metastatic prostate macrophage-autologous human granulocytes H transduced with the prostate cancer vaccine 03/8/94 *
*(primer protocolo que va a aprobarse bajo un proceso acelerado de revisión; ORDA = Oficina de* (first protocol to be approved under an accelerated review process; ORDA = Office of
Actividades con ADN Recombinante)Activities with Recombinant DNA)
Cáncer de mama Infección in vivo con el vector retroviral dirigido a Ia 12/9/94 metastático mama que expresa ARN antisentido de c-fos o antisentido de c-mycBreast cancer In vivo infection with the retroviral vector directed to the 12/9/94 metastatic breast that expresses c-fos antisense or c-myc antisense RNA
Cáncer de mama Sistema no viral (basado en liposomas) para 12/9/94 metastásico (que no suministrar el gen de interleucina-2 humano a responde al tratamiento o células tumorales autólogas (estudio piloto) recurrente) Síndrome de Hunter Transferencia génica mediada por retrovirales del 13/9/94 leve gen de Ia iduronato-2-sulfatasa a linfocitosBreast cancer Non-viral system (based on liposomes) for 12/9/94 metastatic (which does not supply the human interleukin-2 gene to respond to treatment or autologous tumor cells (pilot study) recurrent) Hunter syndrome Gene transfer mediated by 13/9/94 mild retroviral lymphocyte iduronate-2-sulfatase gene
Mesotelioma Uso de adenovirus recombinante (estudio de Fase I) 13/9/94 avanzadoMesothelioma Use of recombinant adenovirus (Phase I study) 9/13/94 advanced
La tabla 1 sólo representa ejemplos de genes que pueden suministrarse según los métodos de Ia invención. En Ia bibliografía, hay publicados promotores, potenciadores, vectores, etc., para tales genes, asociados con los ensayos anteriores. En general, los genes útiles sustituyen o complementan Ia función, incluyendo genes que codifican para enzimas que faltan tales como adenosina desaminasa (ADA) que se ha utilizado en ensayos clínicos para tratar Ia deficiencia de ADA y cofactores tales como insulina y el factor de coagulación VIII. También pueden administrarse genes que afectan a Ia regulación, solos o en combinación con un gen que complementa o sustituye una función específica. Por ejemplo, puede administrarse un gen que codifica para una proteína que suprime Ia expresión de un gen que codifica para una proteína particular.Table 1 only represents examples of genes that can be supplied according to the methods of the invention. In the literature, there are published promoters, enhancers, vectors, etc., for such genes, associated with the previous tests. In general, useful genes substitute or complement the function, including genes that code for missing enzymes such as adenosine deaminase (ADA) that has been used in clinical trials to treat ADA deficiency and cofactors such as insulin and coagulation factor VIII. You can also administer genes that affect regulation, alone or in combination with a gene that complements or replaces a specific function For example, a gene that codes for a protein that suppresses the expression of a gene that codes for a particular protein can be administered.
La invención contempla aún adicionalmente Ia selección de varios compuestos por sus efectos sobre las CMEh proporcionadas en el presente documento. Los compuestos pueden seleccionarse por su capacidad para mantener las líneas de CMEh en un estado no diferenciado, o para inducir Ia diferenciación de las CMEh, o modificar de otro modo las células. Algunos aspectos de Ia selección pueden dirigirse a probar Ia eficacia terapéutica de compuestos candidatos. Dependiendo de los compuestos particulares que se estén seleccionando, variarán las lecturas de los ensayos. Por ejemplo, en algunos ensayos, Ia lectura será el mantenimiento y/o aumento del número de CMEh mientras que en otros Ia lectura será Ia producción de progenie diferenciada (opcionalmente con una disminución concomitante del número de CMEh). También puede seguirse Ia diferenciación de las CMEh a través de cambios en los perfiles de expresión. Por ejemplo, puede identificarse Ia diferenciación de las CMEh mediante Ia regulación por disminución de Ia expresión de SSEA-3 y SSEA-4 y Ia regulación por incremento de Ia expresión de SSEA-1. Aún otros ensayos pueden incluir una lectura de viabilidad celular o alternativamente de muerte celular. Tales ensayos pueden proporcionar entonces lecturas in vitro que se correlacionan potencialmente con Ia toxicidad y/o eficacia que los compuestos de prueba mostrarían en sujetos humanos. Por tanto, el efecto del agente sobre Ia línea de CMEh o su progenie diferenciada in vitro es una forma de marcador o lectura sustituto sobre cómo funcionará el agente ¡n vivo. Las líneas pueden utilizarse adicionalmente como un sistema modelo en el que desarrollar un régimen terapéutico personalizado para un paciente que puede ser genéticamente similar o histocompatible con Ia línea.The invention further contemplates the selection of various compounds for their effects on the CMEhs provided herein. The compounds can be selected for their ability to maintain the CMEh lines in an undifferentiated state, or to induce the differentiation of the CMEh, or otherwise modify the cells. Some aspects of the selection may be directed to test the therapeutic efficacy of candidate compounds. Depending on the particular compounds being selected, test readings will vary. For example, in some tests, the reading will be the maintenance and / or increase in the number of CMEh while in others the reading will be the production of differentiated progeny (optionally with a concomitant decrease in the number of CMEh). The differentiation of the CMEh can also be followed through changes in the expression profiles. For example, the differentiation of the CMEh can be identified by the regulation by decrease of the expression of SSEA-3 and SSEA-4 and the regulation by increase of the expression of SSEA-1. Still other tests may include a reading of cell viability or alternatively cell death. Such tests can then provide in vitro readings that potentially correlate with the toxicity and / or efficacy that the test compounds would show in human subjects. Therefore, the effect of the agent on the CMEh line or its differentiated progeny in vitro is a form of marker or substitute reading on how the live agent will work. The lines can be used additionally as a model system in which to develop a personalized therapeutic regimen for a patient that can be genetically similar or histocompatible with the line.
La presente invención se ilustra adicionalmente mediante los siguientes ejemplos, que no deben considerarse en modo alguno como adicionalmente limitantes. EiemplosThe present invention is further illustrated by the following examples, which should not be considered in any way as additionally limiting. Examples
Derivación de líneas de CMEhCMEh line derivation
Las primeras dos líneas de células madre embrionarias humanas (VAL-1 y VAL-2) se han derivado en España con embriones crioconservados a largo plazo en condiciones libres de animales. Todos los embriones congelados durante > 5 años fueron donados tras el consentimiento informado para Ia derivación de células madre, según Ia ley española 45/2003 del 21 noviembre de 2003. Se descongelaron cuarenta embriones humanos que se habían crioconservado en el día 2 de desarrollo durante > 5 años. Seis embriones no sobrevivieron a este proceso: 5 se degeneraron y se fracturó Ia zona de 1 embrión. Se detuvo un total de 15 de 34 embriones (44,1%) durante las fases iniciales de desarrollo: 12 se detuvieron su crecimiento el primer día (35,3%) y 3 (8,8%) posteriormente. Adicionalmente, se formaron 3 pseudoblastocistos (8,8%). En total, se obtuvieron 16 blastocistos (47,1%) y se clasificaron según Gardner et al. Fértil Steril 1998 69:84-88. De ellos, 11 blastocistos (68,7%) tuvieron un grado de masa celular interna (MCI) de o bien A o bien B.The first two human embryonic stem cell lines (VAL-1 and VAL-2) have been derived in Spain with long-term cryopreserved embryos in animal-free conditions. All frozen embryos for> 5 years were donated after informed consent for stem cell bypass, according to Spanish law 45/2003 of November 21, 2003. Forty human embryos that had been cryopreserved on day 2 of development were thawed. > 5 years. Six embryos did not survive this process: 5 degenerated and the area of 1 embryo fractured. A total of 15 of 34 embryos (44.1%) were stopped during the initial stages of development: 12 growth was stopped on the first day (35.3%) and 3 (8.8%) subsequently. Additionally, 3 pseudoblasts were formed (8.8%). In total, 16 blastocysts (47.1%) were obtained and classified according to Gardner et al. Fertile Steril 1998 69: 84-88. Of these, 11 blastocysts (68.7%) had a degree of internal cell mass (MCI) of either A or B.
Se ha descrito una descripción completa de Ia producción de líneas de fibroblastos placentarios por Genbacev et al. Fértil Steril 2005 83:1517-1529. Los fibroblastos placentarios humanos obtenidos a partir de placentas humanas de gestación temprana apoyan Ia propagación de líneas establecidas de CMEh. En todos los aspectos, los fibroblastos placentarios fueron comparables a los fibroblastos de ratón como células alimentadoras de CMEh. Posteriormente, se utilizó una línea de fibroblastos placentarios cualificados como una capa alimentadora para Ia derivación de nuevas CMEh. Brevemente, se eliminó Ia zona pelúcida con solución acida de Tyrode tal como se describe en Genbacev et al., y los blastocistos (véanse las figuras 1A y D) se sembraron en placa sobre las células alimentadoras formadas a partir de fibroblastos placentarios humanos irradiados en medio definido: 79% de medio esencial mínimo de Dulbecco (DMEM) deficiente (Gibco/BRL, Paisley, Reino Unido), 20% de suero recombinante (SR) deficiente (Gibco/BRL), glutamina 1 mmol/L (Gibco/BRL), ^-mercaptoetanol 0,1 mmol/L (Sigma, St. Louis, MO), 1 % de reserva de aminoácidos no esenciales (Gibco/BRL), que contiene factor de crecimiento de fibroblastos básico humano 12 ng/mL (Invitrogen; Life Technologies, Carlsbad, CA). El proceso de derivación se llevó a cabo según las BPF.A complete description of the production of placental fibroblast lines has been described by Genbacev et al. Fertile Steril 2005 83: 1517-1529. Human placental fibroblasts obtained from human placentas of early gestation support the propagation of established CMEh lines. In all aspects, placental fibroblasts were comparable to mouse fibroblasts as feeder cells for CMEh. Subsequently, a line of qualified placental fibroblasts was used as a feeder layer for the derivation of new CMEh. Briefly, the zona pellucida with Tyrode acid solution was removed as described in Genbacev et al., And the blastocysts (see Figures 1A and D) were plated on feeder cells formed from irradiated human placental fibroblasts in Defined medium: 79% of Dulbecco's minimum essential medium (DMEM) deficient (Gibco / BRL, Paisley, United Kingdom), 20% deficient recombinant serum (SR) (Gibco / BRL), 1 mmol / L glutamine (Gibco / BRL ), ^ -mercaptoethanol 0.1 mmol / L (Sigma, St. Louis, MO), 1% non-essential amino acid reserve (Gibco / BRL), which contains 12 ng / mL basic human fibroblast growth factor (Invitrogen; Life Technologies, Carlsbad, CA). The referral process was carried out according to GMP.
En el plazo de los primeros 2 días, 14 blastocistos se unieron (87,5%) a las células alimentadoras humanas, y en dos casos aparecieron crecimientos con morfología similar a CMEh a los 5 - 7 días tras Ia siembra en placa. Dieciocho y 21 días más tarde, se diseccionaron mecánicamente MCI expandidas individuales (véanse las figuras 1 B y E) en tres trozos y se les permitió que volvieran a unirse en el mismo pocilio. Después de 6 - 8 días, las colonias se dividieron de nuevo mecánicamente y luego se transfirieron a células alimentadoras de fibroblastos placentarios humanos nuevos. La derivación satisfactoria se asoció con Ia aparición de células redondas con nucléolos prominentes (véanse las figuras 1C y F), mientras que las células con una morfología de tipo estromal o bien murieron o bien desaparecieron tras los pases. Por tanto hasta ahora, las nuevas líneas de CMEh se han cultivado y diseccionado mecánicamente hasta el pase 120 (VAL-1) y 85 (VAL-2) en las mismas condiciones que se utilizaron para Ia derivación (es decir, sobre células alimentadoras de fibroblastos humanos placentarios en medio definido sin suero). Adicionalmente, VAL-1 y VAL-2 se han crioconservado y descongelado satisfactoriamente utilizando el método de congelación lenta convencional con dimetüsulfóxido al 10% como crioprotector, tal como describen Bongso et al. Hum. Reprod. 11:2110-2117, 1994.Within the first 2 days, 14 blastocysts joined (87.5%) to human feeder cells, and in two cases there were growths with morphology similar to CMEh at 5-7 days after plating. Eighteen and 21 days later, individual expanded MCIs (see Figures 1 B and E) were mechanically dissected into three pieces and allowed to rejoin in the same well. After 6-8 days, the colonies were mechanically divided again and then transferred to feeder cells of new human placental fibroblasts. The satisfactory derivation was associated with the appearance of round cells with prominent nucleoli (see Figures 1C and F), while the cells with a stromal morphology either died or disappeared after the passes. Therefore, until now, the new CMEh lines have been mechanically cultivated and dissected up to pass 120 (VAL-1) and 85 (VAL-2) under the same conditions that were used for the derivation (that is, on feeder cells of placental human fibroblasts in defined medium without serum). Additionally, VAL-1 and VAL-2 have been cryopreserved and thawed successfully using the conventional slow freezing method with 10% dimethylsulfoxide as a cryoprotectant, as described by Bongso et al. Hum. Play 11: 2110-2117, 1994.
Estos datos sugieren una eficacia de derivación del 5% por embrión congelado o del 12,5% por blastocisto.These data suggest a shunt efficiency of 5% by frozen embryo or 12.5% by blastocyst.
Caracterización de líneas de CMEhCMEh line characterization
Tras los pases, las colonias de VAL-1 y VAL-2 tenían un área superficial mayor, parecían más delgadas y más planas, y tenían límites definidos rectos, proporcionando a las colonias bordes angulares o circulares (véanse las figuras 1 C y F). Con un alto aumento, las CMEh individuales sobre las células alimentadoras humanas eran pequeñas y redondas, con nucléolos prominentes, una característica típica de estas células. Comenzando en el pase 4, se obtuvo el cariotipo de las células cada vez que se dividieron las colonias. Se incubaron las CMEh en medio hES, se complementaron con colcemida 0,2 μg/ml (ROCHE, disolución madre de 10 μg/ml) a 370C durante 30 minutos, y posteriormente se lavaron tres veces con 2 mi de PBS +Ca +Mg. Se diseccionó mecánicamente un mínimo de 20 colonias sobre PBS a partir de Ia capa alimentadora, se recogieron en 2 mi de 1x tripsina-EDTA y se incubaron a 370C durante 5 minutos. La mezcla final de células se pipeteó varias veces al final de Ia incubación, con el fin de garantizar Ia disgregación total en células individuales. Se detuvo Ia actividad tripsina con 4 mi de medio hES y se centrifugó a 1800 rpm durante 10 minutos. Después de eso, se desechó el sobrenadante y se resuspendió cuidadosamente el sedimento y se incubó en 1 mi de disolución de cloruro de potasio precalentada (KCI, 0,075 M) durante 10 minutos a 370C. Las células se prefijaron con 1 mi de disolución fijadora de Camoy (3 vols. de metanol: 1 vol. de ácido acético) a - 2O0C, y se centrifugó inmediatamente a 1800 rpm durante 10 minutos. Finalmente, se desechó el sobrenadante y se resuspendió de nuevo en disolución de Camoy. Se evaluaron análisis citogenéticos de al menos 20 distribuciones de metafase y cinco cariotipos en bandas para determinar las redisposiciones cromosómicas utilizando el método de bandas GTG por dos genetistas cualificados en Prenatal Genetics (Barcelona, España). Cada análisis mostró que ambas líneas celulares mantuvieron un cariotipo 46, XX normal (véanse las figuras 2A y F).After the passes, the colonies of VAL-1 and VAL-2 had a larger surface area, appeared thinner and flatter, and had straight defined boundaries, providing the colonies with angular or circular edges (see Figures 1 C and F) . With a high increase, the individual CMEh on human feeder cells were small and round, with prominent nucleoli, a typical characteristic of these cells. Starting at pass 4, the karyotype of the cells was obtained each time the colonies were divided. HESCs were incubated in hES, supplemented with colcemid 0.2 .mu.g / ml (ROCHE, stock solution 10 mg / ml) at 37 0 C for 30 minutes, and subsequently washed three times with 2 mi PBS + Ca of + Mg A minimum of 20 colonies on PBS were mechanically dissected from Ia feeder layer, they were collected in 2 ml of 1x trypsin-EDTA and incubated at 37 0 C for 5 minutes. The final mixture of cells was pipetted several times at the end of the incubation, in order to guarantee total disintegration in individual cells. Trypsin activity was stopped with 4 ml of hES medium and centrifuged at 1800 rpm for 10 minutes. After that, the supernatant was discarded and the sediment was carefully resuspended and incubated in 1 ml of preheated potassium chloride solution (KCI, 0.075 M) for 10 minutes at 37 ° C. The cells were pre-fixed with 1 ml of solution. Camoy fixative (3 vols of methanol: 1 vol. of acetic acid) at - 2O 0 C, and was immediately centrifuged at 1800 rpm for 10 minutes. Finally, the supernatant was discarded and resuspended again in Camoy's dissolution. Cytogenetic analyzes of at least 20 metaphase distributions and five band karyotypes were evaluated to determine chromosomal redispositions using the GTG band method by two qualified geneticists at Prenatal Genetics (Barcelona, Spain). Each analysis showed that both cell lines maintained a normal 46, XX karyotype (see Figures 2A and F).
Las colonias de CMEh fijadas se expusieron a anticuerpos primarios específicos para antígenos de rechazo tumoral TRA-1-60 y TRA-1-81 (facilitados generosamente por Peter Andrews, Sheffield University), y antígeno embrionario específico de fase 4 (SSEA-4), (Chemícon, Temecula, CA). Se demostró Ia actividad fosfatasa alcalina mediante un kit de sustrato de vector Blue/Red (Vector Laboratories, Burlingame, CA). Los estudios de inmunolocalización mostraron que VAL-1 y VAL-2 expresaron SSEA-4 (Chemicon; Temecula, CA) (véanse las figuras 2B y G), TRA-1-60 (véanse las figuras 2C y H) y TRA-1-81 (véanse las figuras 2D y I). También se detectó actividad fosfatasa alcalina (véanse las figuras 2E y J). También se realizó PCR a tiempo real de varios marcadores. Se extrajo ARN total de colonias de VAL-1 y VAL-2 de grado A utilizando el reactivo TRIzol (Invitrogen) según las instrucciones del fabricante para cantidades de células a pequeña escala, y se evaluó Ia concentración de ARN utilizando un espectrofotómetro (BioRad). Se utilizó ARN total (1 μg) de cada muestra para una síntesis de ADNc de primera hebra utilizando el kit Advantage RT para PCR (BD Biosciences) siguiendo el protocolo del fabricante. Se diseñaron cebadores para PCR utilizando el software basado en Ia red disponible de manera gratuita (Primer3, Genefisher). Se llevaron a cabo reacciones de PCR que utilizan 1 μg de ADNc total como molde, tal como sigue: desnaturalización a 940C durante 4 minutos, y ciclos 40 veces a 940C durante 1 minuto, 550C durante 1 minuto y 72 0C durante 1 minuto. Se realizó una extensión final a 720C durante 10 minutos tras los ciclos. Se resolvieron los productos de PCR en geles de agarosa al 2% se tiñeron con bromuro de etidio, y se visualizaron en un transiluminador (BioRad). Estos estudios de RT-PCR demostraron que ambas líneas celulares eran positivas para Oct-4, Rex-1 , Cripto, Thy-1 , y Nanog y eran negativas para marcadores de diferenciación clásicos: Matni , Amylase, y Dbh.The fixed CMEh colonies were exposed to specific primary antibodies to tumor rejection antigens TRA-1-60 and TRA-1-81 (generously provided by Peter Andrews, Sheffield University), and phase 4 specific embryonic antigen (SSEA-4) , (Chemícon, Temecula, CA). The alkaline phosphatase activity was demonstrated by means of a Blue / Red vector substrate kit (Vector Laboratories, Burlingame, CA). Immunolocation studies showed that VAL-1 and VAL-2 expressed SSEA-4 (Chemicon; Temecula, CA) (see Figures 2B and G), TRA-1-60 (see Figures 2C and H) and TRA-1 -81 (see Figures 2D and I). Alkaline phosphatase activity was also detected (see Figures 2E and J). Real-time PCR of several markers was also performed. Total RNA from VAL-1 and VAL-2 grade A colonies was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions for small-scale cell quantities, and the RNA concentration was evaluated using a spectrophotometer (BioRad) . Total RNA (1 μg) of each sample was used for a first strand cDNA synthesis using the Advantage RT PCR kit (BD Biosciences) following the manufacturer's protocol. PCR primers were designed using the network-based software available for free (Primer3, Genefisher). PCR reactions were carried out using 1 μg of total cDNA as a template, as follows: denaturation at 94 0 C for 4 minutes, and cycles 40 times at 94 0 C for 1 minute, 55 0 C for 1 minute and 72 0 C for 1 minute. A final extension was performed at 72 0 C for 10 minutes after the cycles. The PCR products were resolved in 2% agarose gels, stained with ethidium bromide, and visualized on a transilluminator (BioRad). These RT-PCR studies showed that both cell lines were positive for Oct-4, Rex-1, Crypto, Thy-1, and Nanog and were negative for classic differentiation markers: Matni, Amylase, and Dbh.
Se analizó Ia actividad telomerasa de VAL-1 y VAL-2 utilizando un kit de detección de telomerasa TRAPEZE ® (Chemicon) seguido por tinción con SYBR® (Molecular Probes, " Eugene, OR). Brevemente, se recogieron las colonias (50-100) se lavaron una vez con PBS libre de Ca++ y Mg++ y se resuspendieron inmediatamente en tampón de lisis. Tras tratamiento en hielo y centrifugación, las muestras se sometieron a una reacción de PCR siguiendo las instrucciones del fabricante. Los productos de PCR se sometieron a ensayo en un gel de poliacrilamida (15% de TBE, BioRad) en condiciones no desnaturalizantes, y los fragmentos amplificados se tiñeron con verde SYBR para su visualización en un transiluminador. Los controles negativos se obtuvieron mediante inactivación térmica de las líneas. El producto final contenía una escala de productos de amplificación con incrementos de 6 pb comenzando en 50 pb (véase Ia figura 3). La banda de 36 pb corresponde al control interno de Ia reacción en cadena de Ia polimerasa y Ia banda de 50 pb, a Ia actividad telomerasa que aumenta en bandas de 6 pb en las células inmortales.The telomerase activity of VAL-1 and VAL-2 was analyzed using a TRAPEZE ® (Chemicon) telomerase detection kit followed by staining with SYBR ® (Molecular Probes, " Eugene, OR). Briefly, colonies were collected (50- 100) washed once with PBS free of Ca ++ and Mg ++ and immediately resuspended in lysis buffer After ice treatment and centrifugation, the samples were subjected to a PCR reaction following the manufacturer's instructions. PCR were tested on a polyacrylamide gel (15% TBE, BioRad) under non-denaturing conditions, and the amplified fragments were stained with SYBR green for visualization in a transilluminator.The negative controls were obtained by thermal inactivation of the The final product contained a scale of amplification products with increments of 6 bp starting at 50 bp (see Figure 3) The band of 36 bp corresponds to the internal control of the reaction in polymerase chain and The 50 bp band, at the telomerase activity that increases in 6 bp bands in the immortal cells.
El análisis adicional de VAL-1 y VAL-2 reveló que las líneas eran inmunológicamente idénticas para los marcadores de HLA, A2, A23, B44, B40, CW4, CW5, DR7, DR15, DQ2 y DQ6. Este nivel de histocompatibilidad indica que las líneas pueden utilizarse conjuntamente en un régimen terapéutico con poco o ningún riesgo de rechazo inmunológico. Sin embargo, las líneas no son genéticamente idénticas, tal como se muestra en las figuras 4, 5 y 6.Further analysis of VAL-1 and VAL-2 revealed that the lines were immunologically identical for the markers of HLA, A2, A23, B44, B40, CW4, CW5, DR7, DR15, DQ2 and DQ6. This level of histocompatibility indicates that the lines can be used together in a therapeutic regimen with little or no risk of immune rejection. However, the lines are not genetically identical, as shown in Figures 4, 5 and 6.
VAL-1 y VAL-2 también se evaluaron para determinar sus perfiles de diferenciación espontánea. Se disociaron colonias mediante tratamiento con colagenasa IV durante 5 minutos a 370C y después se cultivaron en suspensión en placas de cultivo de 6 pocilios de escasa unión. El medio de diferenciación consistió en 80% de DMEM, 20% de FBS (Hyclone), L-glutamina 1 mM, β- mercaptoetanol 0,1 mM, y MEM 1 mM aminoácidos no esenciales. Tras 4 días en suspensión, los cuerpos embrioides se transfirieron en cámaras de cultivo recubiertas con poli-L-ornitina y se cultivaron durante 10 - 14 días adicionales. Los cultivos se fijaron con paraformaldehído al 4% durante 20 minutos antes de los análisis de inmunolocalización. Para evaluar Ia expresión de los marcadores asociados con Ia diferenciación de las tres capas germinativas, los cuerpos embrioides se incubaron con, por ejemplo, anti-σ-fetoproteína (ascitis) de ratón (diluida 1 :500; Sigma), anti-β-tubulina III (ascitis) de ratón (diluida 1 :1 ,000; Sigma), o anti-actina del músculo liso de ratón (10,7 μg/mL; Dako). Los controles negativos incluyeron Ia omisión de los anticuerpos primarios y Ia incubación con una IgG no específica. VAL-1 mostró Ia capacidad para diferenciarse espontáneamente en células similares a los miocardiocitos (figuras 7A-D), y tanto VAL-1 como VAL-2 mostraron Ia capacidad de diferenciarse espontáneamente en células similares a neuronas (figura 8B), y células similares al músculo (Ia figura 8C).VAL-1 and VAL-2 were also evaluated to determine their spontaneous differentiation profiles. Colonies were dissociated by collagenase IV treatment for 5 minutes at 37 0 C and then grown in suspension culture plates 6 wells of poor binding. The differentiation medium consisted of 80% DMEM, 20% FBS (Hyclone), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and MEM 1 mM non-essential amino acids. After 4 days in suspension, the embryoid bodies were transferred in culture chambers coated with poly-L-ornithine and cultured for an additional 10-14 days. Cultures were fixed with 4% paraformaldehyde for 20 minutes before immunolocation analyzes. To evaluate the expression of the markers associated with the differentiation of the three germinative layers, the embryoid bodies were incubated with, for example, mouse anti-σ-fetoprotein (ascites) (diluted 1: 500; Sigma), anti-β- mouse tubulin III (ascites) (diluted 1: 1, 000; Sigma), or anti-actin of the mouse smooth muscle (10.7 μg / mL; Dako). Negative controls included the omission of the primary antibodies and the incubation with a non-specific IgG. VAL-1 showed the ability to differentiate spontaneously in cells similar to myocardiocytes (Figures 7A-D), and both VAL-1 and VAL-2 showed the ability to differentiate spontaneously in cells similar to neurons (Figure 8B), and similar cells to the muscle (Figure 8C).
También se analizaron los perfiles de expresión génica de Ia progenie diferenciada espontáneamente de las líneas. Tal como se muestra en Ia tabla 2, las líneas pueden expresar espontáneamente genes asociados con linajes de endodermo, mesodermo y ectodermo. Tabla 2. Perfil de expresión génica de Ia diferenciación espontáneaThe gene expression profiles of the spontaneously differentiated progeny of the lines were also analyzed. As shown in Table 2, the lines can spontaneously express genes associated with endoderm, mesoderm and ectoderm lineages. Table 2. Gene expression profile of spontaneous differentiation.
Figure imgf000033_0001
Figure imgf000033_0001
"¿" indica resultados no concluyentes"¿" Indicates inconclusive results
También se evaluó Ia capacidad de las líneas para formar teratomas in vivo. Se administraron 2x 105 CMEh no diferenciadas mediante inyección intratesticular en ratones CB17 con deficiencia inmunitaria combinada grave (SCID) (n = 22). Los tejidos se recogieron tras 11 semanas y se llevaron a cabo análisis histológicos. Ambas líneas formaron teratomas en los ratones receptores. Un ejemplo de un teratoma resultante y su análisis histológico se muestran en las figuras 9A-D y 10A-L. Estos análisis demuestran Ia capacidad de las líneas para diferenciarse en uno o más linajes de endodermo (por ejemplo, epitelio intestinal, etc.), o uno o más de ectodermo (por ejemplo, rosetas neuronales, etc.) y uno o más de mesodermo (por ejemplo, cartílago, etc.).The ability of the lines to form teratomas in vivo was also evaluated. 2 x 10 5 CMEh undifferentiated were administered by intratesticular injection in CB17 mice with severe combined immune deficiency (SCID) (n = 22). Tissues were collected after 11 weeks and histological analyzes were performed. Both lines formed teratomas in recipient mice. An example of a resulting teratoma and its histological analysis are shown in Figures 9A-D and 10A-L. These analyzes demonstrate the ability of the lines to differentiate into one or more endoderm lineages (for example, intestinal epithelium, etc.), or one or more of ectoderm (for example, neural rosettes, etc.) and one or more mesoderm (for example, cartilage, etc.).
EquivalentesEquivalent
Debe entenderse que Io anterior es simplemente una descripción detallada de ciertas realizaciones. Por tanto, debe ser evidente para el experto habitual en Ia técnica que pueden realizarse diversas modificaciones y equivalentes sin apartarse del espíritu y el alcance de Ia invención, y con no más que experimentación de rutina. Toda Ia bibliografía, patentes y solicitudes de patente que se citan en esta solicitud se incorporan mediante referencia en el presente documento en su totalidad: It should be understood that the above is simply a description detailed of certain embodiments. Therefore, it should be evident to the person skilled in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention, and with no more than routine experimentation. All bibliography, patents and patent applications cited in this application are incorporated by reference in this document in its entirety:

Claims

Reivindicaciones Claims
1. Población aislada de células madre embrionarias humanas en Ia que dicha población de células (1) tiene un cariotipo normal tras al menos 85 pases; (2) expresa el antígeno embrionario específico de fase (SSEA) 4, el antígeno de rechazo tumoral (TRA)-I -60 y TRA-1-81; (3) es positiva para fosfatasa alcalina; (4) expresa Oct-4, Rex-1 , Cripto, Thy-1 y Nanog; (5) es negativa para Matni , Amylase y Dbh; y (6) tiene actividad telomerasa.1. Population isolated from human embryonic stem cells in which said population of cells (1) has a normal karyotype after at least 85 passes; (2) expresses the phase-specific embryonic antigen (SSEA) 4, the tumor rejection antigen (TRA) -I-60 and TRA-1-81; (3) is positive for alkaline phosphatase; (4) expresses Oct-4, Rex-1, Crypto, Thy-1 and Nanog; (5) is negative for Matni, Amylase and Dbh; and (6) has telomerase activity.
2. Población aislada de células madre embrionarias humanas en Ia que dicha población de células es VAL-1 , y Ia progenie de Ia misma.2. Population isolated from human embryonic stem cells in which said population of cells is VAL-1, and the progeny thereof.
3. Población aislada de células madre embrionarias humanas en Ia que dicha población de células es VAL-2, y Ia progenie de Ia misma.3. Population isolated from human embryonic stem cells in which said population of cells is VAL-2, and the progeny thereof.
4. Población aislada de células madre embrionarias humanas en Ia que dicha población de células tiene las características de Ia población de Ia reivindicación 2.4. Population isolated from human embryonic stem cells in which said population of cells has the characteristics of the population of claim 2.
5. Población aislada de células madre embrionarias humanas en Ia que dicha población de células tiene las características de Ia población de Ia reivindicación 3.5. Population isolated from human embryonic stem cells in which said population of cells has the characteristics of the population of claim 3.
6. Población aislada de células madre embrionarias humanas según Ia reivindicación 1 , 4 o 5, en Ia que Ia población comprende células modificadas genéticamente.6. Population isolated from human embryonic stem cells according to claim 1, 4 or 5, wherein the population comprises genetically modified cells.
7. Población aislada de células madre embrionarias humanas según Ia reivindicación 1-4 o 5, en Ia que Ia población se somete a pases al menos 50 veces.7. Population isolated from human embryonic stem cells according to claim 1-4 or 5, in which the population is subjected to passes at least 50 times.
8. Población aislada de células madre embrionarias humanas según Ia reivindicación 1-4 o 5, en Ia que Ia población se somete a pases al menos 75 veces.8. Population isolated from human embryonic stem cells according to claim 1-4 or 5, in which the population is subjected to passes at least 75 times.
9. Población aislada de células madre embrionarias humanas según Ia reivindicación 1-4 ó 5, en Ia que Ia población se somete a pases al menos 100 veces.9. Population isolated from human embryonic stem cells according to claim 1-4 or 5, in which the population is subjected to passes at least 100 times.
10. Población aislada de células madre embrionarias humanas según Ia reivindicación 1-4 ó 5, en Ia que Ia población se crioconserva.10. Population isolated from human embryonic stem cells according to claim 1-4 or 5, in which the population is cryopreserved.
11. Población aislada de células madre embrionarias humanas según11. Population isolated from human embryonic stem cells according to
Ia reivindicación 1-4 ó 5, en Ia que Ia población se crioconserva durante más de un año.The claim 1-4 or 5, wherein the population is cryopreserved for more than one year.
12. Población aislada de células madre embrionarias humanas en Ia que Ia población es una versión modificada genéticamente de Ia línea de células madre embrionarias humanas VAL-1.12. Population isolated from human embryonic stem cells in which the population is a genetically modified version of the human embryonic stem cell line VAL-1.
13. Población aislada de células madre embrionarias humanas en Ia que Ia población es una versión modificada genéticamente de Ia línea de células madre embrionarias humanas VAL-2.13. Population isolated from human embryonic stem cells in which the population is a genetically modified version of the human embryonic stem cell line VAL-2.
14. Cultivo que comprende Ia población de células madre embrionarias humanas según Ia reivindicación 1-12 ó 13.14. Culture comprising the population of human embryonic stem cells according to claim 1-12 or 13.
15. Cultivo según Ia reivindicación 14, que comprende además células alimentadoras humanas.15. Culture according to claim 14, further comprising human feeder cells.
16. Cultivo según Ia reivindicación 15, en el que las células alimentadoras humanas son células alimentadoras placentarias humanas.16. Culture according to claim 15, wherein the human feeder cells are human placental feeder cells.
17. Cultivo según Ia reivindicación 14, en el que el cultivo es un cultivo libre de suero. 17. Culture according to claim 14, wherein the culture is a serum free culture.
18. Cultivo según Ia reivindicación 14, que comprende además el factor de crecimiento de fibroblastos (FGF).18. Culture according to claim 14, further comprising the fibroblast growth factor (FGF).
19. Cultivo según Ia reivindicación 18, en el que el FGF es FGF básico.19. Culture according to claim 18, wherein the FGF is basic FGF.
20. Cultivo según Ia reivindicación 14, en el que el cultivo está libre de células alimentadoras.20. Culture according to claim 14, wherein the culture is free of feeder cells.
21. Cultivo según Ia reivindicación 15, en el que las células alimentadoras placentarias humanas se inactivan mitóticamente por irradiación.21. Culture according to claim 15, wherein the human placental feeder cells are inactivated mitotically by irradiation.
22. Método para cultivar una población de células madre embrionarias humanas que comprende cultivar Ia población de células madre embrionarias humanas según Ia reivindicación 1-12 o 13 en presencia de células alimentadoras humanas en un medio libre de suero.22. Method for cultivating a population of human embryonic stem cells comprising culturing the population of human embryonic stem cells according to claim 1-12 or 13 in the presence of human feeder cells in a serum-free medium.
23. Método según Ia reivindicación 22, en el que el medio comprende23. Method according to claim 22, wherein the medium comprises
FGF.FGF
24. Método según Ia reivindicación 23, en el que FGF es FGFb.24. Method according to claim 23, wherein FGF is FGFb.
25. Método según Ia reivindicación 24, en el que el FGFb está presente en una cantidad de aproximadamente 1-15 ng/mL25. Method according to claim 24, wherein the FGFb is present in an amount of approximately 1-15 ng / mL
26. Método según Ia reivindicación 22, en el que las células alimentadoras humanas son células alimentadoras placentarias humanas.26. Method according to claim 22, wherein the human feeder cells are human placental feeder cells.
27. Método según Ia reivindicación 26, en el que las células alimentadoras placentarias humanas se inactivan mitóticamente por irradiación. 27. Method according to claim 26, wherein the human placental feeder cells are inactivated mitotically by irradiation.
28. Método para diferenciar in vitro una población de células madre embrionarias humanas que comprende exponer a Ia población de células madre embrionarias humanas según Ia reivindicación 1-12 o 13 a condiciones de diferenciación durante un tiempo suficiente para permitir Ia diferenciación de Ia población de células madre embrionarias humanas en células diferenciadas.28. Method for differentiating in vitro a population of human embryonic stem cells comprising exposing the population of human embryonic stem cells according to claim 1-12 or 13 to differentiation conditions for a time sufficient to allow differentiation of the population of cells Human embryonic mother in differentiated cells.
29. Método según Ia reivindicación 28, en el que las condiciones de diferenciación comprenden una condición libre de células alimentadoras.29. Method according to claim 28, wherein the differentiation conditions comprise a condition free of feeder cells.
30. Método según Ia reivindicación 28, en el que las condiciones de diferenciación comprenden células alimentadoras.30. Method according to claim 28, wherein the differentiation conditions comprise feeder cells.
31. Método según Ia reivindicación 28, en el que las condiciones de diferenciación comprenden uno o más factores seleccionados del grupo que consiste en ácido retinoico, factor de crecimiento epidérmico (EGF), proteína morfogenética ósea 4 (BMP4), factor de crecimiento de fibroblastos (FGF), hormonas esteroideas, activina-A, factor de crecimiento transformante beta 1 (TGF-/51), factor de crecimiento de hepatocitos (HGF), y factor de crecimiento nervioso (NGF).31. A method according to claim 28, wherein the differentiation conditions comprise one or more factors selected from the group consisting of retinoic acid, epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), fibroblast growth factor (FGF), steroid hormones, activin-A, transforming growth factor beta 1 (TGF- / 51), hepatocyte growth factor (HGF), and nerve growth factor (NGF).
32. Método según Ia reivindicación 28, que comprende además introducir las células diferenciadas en un sujeto.32. Method according to claim 28, further comprising introducing the differentiated cells into a subject.
33. Método para diferenciar una población de células madre embrionarias humanas que comprende introducir Ia población de células madre embrionarias humanas de Ia reivindicación 1-4 ó 5 en un sujeto humano.33. Method for differentiating a population of human embryonic stem cells comprising introducing the population of human embryonic stem cells of claim 1-4 or 5 into a human subject.
34. Método según Ia reivindicación 31 , en el que el sujeto tiene un estado que afecta al hígado, músculo, piel, cerebro, sistema nervioso, corazón, sistema circulatorio, sistema hematopoyético, páncreas o hueso.34. Method according to claim 31, wherein the subject has a state that affects the liver, muscle, skin, brain, nervous system, heart, circulatory system, hematopoietic system, pancreas or bone.
35. Método según Ia reivindicación 31 , en el que Ia población se introduce en el sujeto humano mediante administración local.35. Method according to claim 31, wherein the population is introduced into the human subject by local administration.
36. Método según Ia reivindicación 35, en el que Ia administración local es Ia administración a un órgano o tejido.36. Method according to claim 35, wherein the local administration is the administration to an organ or tissue.
37. Método según Ia reivindicación 31 , en el que Ia población se introduce en un sujeto humano mediante administración sistémica.37. Method according to claim 31, wherein the population is introduced into a human subject by systemic administration.
38. Método según Ia reivindicación 37, en el que Ia administración sistémica es administración intravenosa.38. Method according to claim 37, wherein the systemic administration is intravenous administration.
39. Método según Ia reivindicación 31 , que comprende además exponer a Ia población a diferentes estados in vitro antes de Ia introducción en el sujeto humano.39. Method according to claim 31, further comprising exposing the population to different in vitro states before the introduction into the human subject.
40. Método según Ia reivindicación 31 , en el que el sujeto expresa al menos un marcador de HLA seleccionado del grupo que consiste en A2, A23,40. Method according to claim 31, wherein the subject expresses at least one HLA marker selected from the group consisting of A2, A23,
B44, B40, CW4, CW5, DR7, CD15, DQ2 y DQ6.B44, B40, CW4, CW5, DR7, CD15, DQ2 and DQ6.
41. Método según Ia reivindicación 31, en el que el sujeto expresa los marcadores de HLA A2, A23, B44, B40, CW4, CW5, DR7, CD15, DQ2 y DQ6.41. Method according to claim 31, wherein the subject expresses the markers of HLA A2, A23, B44, B40, CW4, CW5, DR7, CD15, DQ2 and DQ6.
42. Método según Ia reivindicación 31 , en el que el sujeto recibe células VAL-1 y VAL-2 o Ia progenie de las mismas.42. Method according to claim 31, wherein the subject receives VAL-1 and VAL-2 cells or the progeny thereof.
43. Composición que comprende una célula madre embrionaria humana de Ia población de células madre embrionarias humanas de Ia reivindicación 1-12 o 13. 43. Composition comprising a human embryonic stem cell of the population of human embryonic stem cells of claim 1-12 or 13.
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