WO2007082963A1 - Lignées de cellules souches embryonnaires humaines et leurs méthodes d'utilisation - Google Patents

Lignées de cellules souches embryonnaires humaines et leurs méthodes d'utilisation 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)
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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/fr
Priority to US11/655,048 priority patent/US20070202595A1/en
Publication of WO2007082963A1 publication Critical patent/WO2007082963A1/fr

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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

L'invention concerne des lignées de cellules souches embryonnaires humaines innovantes. L'invention concerne également des lignées de cellules souches embryonnaires humaines qui sont génétiquement liées et identiques sur le plan immunologique. L'invention concerne également des méthodes permettant de dériver, de diffuser et d'utiliser ces lignées, éventuellement sans recours à des animaux.
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