WO2008145788A1 - Method for isolation of the inner cell mass in mammal blastocysts - Google Patents
Method for isolation of the inner cell mass in mammal blastocysts Download PDFInfo
- Publication number
- WO2008145788A1 WO2008145788A1 PCT/ES2008/000377 ES2008000377W WO2008145788A1 WO 2008145788 A1 WO2008145788 A1 WO 2008145788A1 ES 2008000377 W ES2008000377 W ES 2008000377W WO 2008145788 A1 WO2008145788 A1 WO 2008145788A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- blastocysts
- isolation
- culture
- cell mass
- blastocyst
- Prior art date
Links
- 210000002459 blastocyst Anatomy 0.000 title claims abstract description 266
- 238000000034 method Methods 0.000 title claims abstract description 137
- 238000002955 isolation Methods 0.000 title claims abstract description 85
- 241000124008 Mammalia Species 0.000 title abstract 2
- 210000004027 cell Anatomy 0.000 claims description 90
- 210000002257 embryonic structure Anatomy 0.000 claims description 54
- 238000000608 laser ablation Methods 0.000 claims description 53
- 108010010803 Gelatin Proteins 0.000 claims description 19
- 239000008273 gelatin Substances 0.000 claims description 19
- 229920000159 gelatin Polymers 0.000 claims description 19
- 235000019322 gelatine Nutrition 0.000 claims description 19
- 235000011852 gelatine desserts Nutrition 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 14
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 12
- 210000004340 zona pellucida Anatomy 0.000 claims description 11
- 238000012423 maintenance Methods 0.000 claims description 10
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 10
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 7
- 210000002950 fibroblast Anatomy 0.000 claims description 7
- 230000008030 elimination Effects 0.000 claims description 6
- 238000003379 elimination reaction Methods 0.000 claims description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- 241000282412 Homo Species 0.000 claims description 5
- 239000003797 essential amino acid Substances 0.000 claims description 5
- 235000020776 essential amino acid Nutrition 0.000 claims description 5
- 230000012447 hatching Effects 0.000 claims description 5
- 210000003101 oviduct Anatomy 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 230000002269 spontaneous effect Effects 0.000 claims description 5
- 229930182816 L-glutamine Natural products 0.000 claims description 4
- 108010059712 Pronase Proteins 0.000 claims description 4
- 230000003287 optical effect Effects 0.000 claims description 4
- 210000002308 embryonic cell Anatomy 0.000 claims description 3
- 208000032839 leukemia Diseases 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 229930182555 Penicillin Natural products 0.000 claims description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 229940049954 penicillin Drugs 0.000 claims description 2
- 241000283690 Bos taurus Species 0.000 claims 1
- 230000001605 fetal effect Effects 0.000 claims 1
- 230000006872 improvement Effects 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000002679 ablation Methods 0.000 abstract 1
- 210000004703 blastocyst inner cell mass Anatomy 0.000 abstract 1
- 101000980673 Homo sapiens Multicilin Proteins 0.000 description 102
- 210000001671 embryonic stem cell Anatomy 0.000 description 62
- 241000699666 Mus <mouse, genus> Species 0.000 description 53
- 238000009795 derivation Methods 0.000 description 37
- 238000005516 engineering process Methods 0.000 description 17
- 230000008569 process Effects 0.000 description 17
- 210000001161 mammalian embryo Anatomy 0.000 description 14
- 230000004069 differentiation Effects 0.000 description 10
- 238000011160 research Methods 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 210000001109 blastomere Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 210000000130 stem cell Anatomy 0.000 description 6
- 241001529936 Murinae Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 208000036878 aneuploidy Diseases 0.000 description 5
- 238000012136 culture method Methods 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000005553 drilling Methods 0.000 description 5
- 230000002708 enhancing effect Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- LAQPKDLYOBZWBT-NYLDSJSYSA-N (2s,4s,5r,6r)-5-acetamido-2-{[(2s,3r,4s,5s,6r)-2-{[(2r,3r,4r,5r)-5-acetamido-1,2-dihydroxy-6-oxo-4-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-3-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-[(1r,2r)-1,2,3-trihydrox Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 LAQPKDLYOBZWBT-NYLDSJSYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 210000002242 embryoid body Anatomy 0.000 description 4
- 230000004720 fertilization Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 210000001654 germ layer Anatomy 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102000008730 Nestin Human genes 0.000 description 3
- 108010088225 Nestin Proteins 0.000 description 3
- 208000012868 Overgrowth Diseases 0.000 description 3
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 3
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 3
- 230000003322 aneuploid effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000002559 cytogenic effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000013401 experimental design Methods 0.000 description 3
- 238000010304 firing Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 210000005055 nestin Anatomy 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101150086694 SLC22A3 gene Proteins 0.000 description 2
- 231100001075 aneuploidy Toxicity 0.000 description 2
- 238000005056 compaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 210000001900 endoderm Anatomy 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000010297 mechanical methods and process Methods 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000000754 repressing effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 244000187656 Eucalyptus cornuta Species 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 238000001358 Pearson's chi-squared test Methods 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007159 enucleation Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- RHCSKNNOAZULRK-UHFFFAOYSA-N mescaline Chemical compound COC1=CC(CCN)=CC(OC)=C1OC RHCSKNNOAZULRK-UHFFFAOYSA-N 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002676 xenobiotic agent Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B17/00—Surgical instruments, devices or methods
- A61B17/42—Gynaecological or obstetrical instruments or methods
- A61B17/425—Gynaecological or obstetrical instruments or methods for reproduction or fertilisation
- A61B17/435—Gynaecological or obstetrical instruments or methods for reproduction or fertilisation for embryo or ova transplantation
-
- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
-
- 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
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
Definitions
- the object of the present invention is a procedure for the isolation of the internal cell mass (MCI) in mammalian blastocysts that includes the direct plate culture of said blastocysts and the subsequent laser ablation of the trophoctoctoder of said blastocysts.
- the process of the invention allows an improvement of the efficiency of the isolation of the internal cell mass of blastocysts and the use of low quality blastocysts not suitable for the separation of its internal cell mass by means of the procedures mentioned in the documents of the state of the art .
- embryonic stem cells were first obtained from mouse blastocysts (Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154-56). These cells are endowed with two unique properties that distinguish them from all organ-specific stem cells identified so far:
- the quality of the blastocysts and the method of isolation of the MCI used are the two main factors that lead to the success or failure of the process of derivation of human embryonic stem cell lines.
- the quality of blastocysts humans are of poorer quality than mouse. This is due, at least in part, to the fact that human blastocysts are frozen when the derivation of human embryonic stem cell lines is attempted, due to ethical restrictions, while mouse blastocysts are normally used fresh. This explains not only the extremely low degree of success of the processes of derivation of human embryonic stem cell lines, but also implies that poor quality human embryos are directly discarded, and even neither defrosted nor used for this purpose.
- MCI is usually isolated from expanded blastocysts using a wide variety of techniques that include immunosurgery (Solter D., Knowles BB. 1975. Immunosurgery of mouse biastocysts; Proc. Nati. Acad. Sci. USA 72: 5099-5102), mechanical procedures (Bongso A, Fong CY, Ng SC, Ratnam S. 1994. Isolation and culture of inner cell mass cells from human biastocysts. Hum. Reprod.
- Laser technology is commonly used in assisted reproduction (Antinori S, Selman HA, Caff B, Panci C, Dani GL, Versad C. 1996. Zone opening of human embryos using non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy Hum. Reprod. 11: 2488-2492), because it facilitates nuclear transfer, accelerating the enucleation process so that it develops in seconds, while avoiding any damage to the ovule (Chen S., Chao K., Chang C 1 Hsieh F., Ho H., Yang Y. 2004.
- Piezo minimizes damage of the ooplasmic membrane at injection. J Exp. Zool. 301: 344-351). Recently, laser ablation technology has been used to destroy the trophoectoderm of good quality mouse blastocysts, allowing to successfully separate the MCI and the subsequent derivation of lines of mouse embryonic stem cells (Cortes JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, Concha A, Menendez P.
- the process object of the present invention allows to overcome these inconveniences, through the combination of a stage of cultivation of the complete blastocysts followed by the destruction of the trophoctoctoder by laser ablation to improve the efficiency of the isolation of the MCI and the derivation of embryonic stem cells from mammalian embryos, specifically mouse and human.
- the object of the present invention constitutes a method for the isolation of MCI in mammalian blastocysts that includes a laser ablation step of the trophoctoctoder of said blastocysts.
- the blastocysts are subjected, previously to said laser ablation stage, to direct plate culture for a period of at least 72 hours.
- the blastocysts are from non-human mammals, particularly mice.
- the direct culture prior to laser ablation includes the following sub-stages: a) culture of the blastocysts recovered from the oviducts in standard medium, particularly G2 V.lll culture medium (Vitrolife, Sweden) at 37 0 C and with 6% CO 2 for a period of time between 24 and 48 hours. b) treatment of the blastocysts cultured in the previous sub-stage to achieve a complete elimination of the zona pellucida. c) placing the blastocysts treated in the previous stage on a culture surface and subsequent adhesion of the trophoctoctoderm and the internal cell mass of the blastocysts to said surface.
- the removal of the zona pellucida is carried out by treatment with Tyrode acid or pronase enzyme for a period of time between 30 and 60 seconds, or by spontaneous hatching of the blastocyst.
- the culture surface on which the blastocysts are placed after the removal of the zona pellucida is a plate covered with MEFs (Mouse embryonic fibroblasts) or with gelatin.
- the medium in which blastocysts are maintained on the surface of the culture is DMEM (Dulbecco 's Modified Eagle Medium) supplemented with fetal bovine serum 20%, L-glutamine , 1% non - essential amino acids 0.1 mM, 2000 lU / ml inhibitory factor Mouse leukemia, 0.1 mM ⁇ -mercaptoethanol, 50 U / ml penicillin and 50 ⁇ g / ml streptomycin.
- the laser ablation stage of the blastocyst trophoectoderm to be performed immediately after the previous culture stage, is carried out with an infrared emission laser diode device at an optical frequency (wavelength) of 1.48 ⁇ m ., taking between 20 and 100 shots.
- an optical frequency (wavelength) of 1.48 ⁇ m ., taking between 20 and 100 shots.
- mammalian blastocysts are human blastocysts.
- the direct culture prior to laser ablation includes the following sub-stages: a) thawing of human embryos in the early division stage and culture until the blastocyst evolution using means G1 and G2 v.5
- the removal of the zona pellucida is carried out by treatment with Tyrode acid or pronase enzyme for a period of time between 30 and 60 seconds or by spontaneous hatching of the blastocyst.
- the surface. of culture on which the blastocysts are placed after the removal of the zona pellucida is a plate covered with "feedérs" human and the environment in which blastocysts on the surface culture are maintained is DMEM (Dulbecco 's Modified Eagle Medium) Knockout (80%), serum replacement (20%), non - essential (1%) amino acids, L -glutamine (1%), 2-mercaptoethanol (0.2%), at 37 ° C and 5% CO 2 .
- DMEM Dulbecco 's Modified Eagle Medium
- the laser ablation stage of the blastocyst trophoectoderm to be performed immediately after the previous culture stage, is carried out with an infrared emission laser diode device at an optical frequency (wavelength) of 1.48 ⁇ m , taking between 20 and 100 shots.
- an optical frequency (wavelength) 1.48 ⁇ m
- Figure 1 Representative figures of the quality classification of the blastocysts obtained.
- MCI internal cell mass
- Figure 2 Experimental design for the optimization of the efficiency of the derivation of mouse embryonic stem cells based on the influence of the quality of the blastocyst, method of isolation of the internal cell mass and growth surface.
- Figure 3 Graphs representative of the assessment of the different methods of isolation of the internal cell mass in mouse blastocysts.
- C-E Isolation of the MCI by means of laser ablation technology.
- G Clean and free MCI of trophoctoctore cells after laser shots.
- H Residual trophoectoderm after laser ablation. Asterisks represent MCIs. The wide white arrows indicate the hairy area of the embryo. The fine white arrows indicate the exact situation of the laser shots.
- Figure 4 Representative graphs showing the derivation achieved and the immunocytochemical characterization of mouse embryonic stem cells, A) Typical morphology (10 X magnification) of embryonic stem cell lines on MEFs established by prior culture of the complete blastocyst and subsequent laser ablation of the trophoctoctoderm.
- Figure 5 Maintenance of the differentiation potential of the three germ layers of mouse embryonic stem cell lines.
- Figure 6 Cytogenetic characterization of established mouse embryonic stem cell lines.
- C High-growth and good-quality blastocyst with a large and clearly distinguishable internal cell mass (MCI). The black arrow indicates where the region of the MCI is located. D. Low growth blastocyst with internal cell mass not distinguishable.
- Figure 8 Experimental design for the optimization of the efficiency of the derivation of human embryonic stem cells based on the influence of the quality of the blastocyst, growth surface and method of isolation of the internal cell mass.
- Figure 9 Representative images of the blastocyst evolution of a thawed human pre-embryo and assessment of the method of isolation of the internal cell mass by direct culture prior to laser ablation.
- the wide white arrows indicate the hairy area of the embryo.
- the fine white arrows indicate the exact situation of the laser shots.
- the blastocysts were obtained by washing the oviducts of mice of the C57BL / CBA strain from 3 to 6 months of age. These mice had been previously sacrificed by cervical dislocation on day 4.5 of pregnancy (counting that the coupling period occurs on day 0.5).
- the oviducts were dissected and placed in PBS medium tempered at 37 0 C and were subsequently carefully handled in a Petri dish with .medio tempered G-MOPS (Vitrolife, Sweden) as described in Tanaka N. et al. "Laser-assisted blastocyst dissection and subsequent; cultivation of embryonic stem cells in a serum / cell free culture system: applications and preliminary results in a murine model" J. Trans ⁇ . Med. 2006; 4: 20-32.
- the oviducts were separated from the portion of the uterus and the ovaries by means of a scalpel section. This procedure was performed with the help of a stereoscopic magnifying glass and an inverted microscope with Hoffman optics.
- the recovered blastocysts were placed in culture medium G2 V.lll (Vitrolife, Sweden) in an incubator at 37 0 C and 6% CO2 as described in the above reference of Tanaka et al. .
- One hundred and eleven mouse blastocysts were included in the present study that provides experimental support to the method of the invention. " They were recovered in the compaction stage or in the expanded blastocyst stage. The blastocysts in the compaction stage were grown to the expanded blastocyst stage.
- blastocysts were classified as blastocysts of good or poor quality: blastocysts of good quality possessed a large and clearly distinguishable MCI. Blastocysts of poor quality were classified when they possessed a small but distinguishable MCI and when they had an indistinguishable MCI.
- the blastocyst was directly cultured in such a way that both Ja MCI and trophoctoctoder cells adhere to MEFs or Ia jelly. Two days later, the blastocyst adhesion to the growth surface was confirmed and the culture medium was changed to fresh tempered medium. On day 3 of culture the cells began to expand providing support for the growth of the MCI by growing and forming a dome-shaped structure. At this time, the MCI was carefully raised and transferred to a fresh growing surface.
- the direct culture method has the risk of overgrowth of the trophoctoctoder, since it is fully cultivated together with the MCI, making it difficult for the embryologist to access the MCI in blastocysts of poor quality.
- the laser system used for the process of the invention (OCTAX EyewareTM, Germany) is connected to an inverted microscope (IX-71, Olympus) and processed by a computer program which allows data to be analyzed.
- the use of the laser consists of firing and lysing the trophoctoctoder cells looking for their separation. This direct firing technique can only be used in mouse blastocysts of good quality, where the MCI is clearly, identifiable and distinguishable from trophoectoderm cells.
- the laser is placed in a certain area of the field located in the target mode sorting screen. The expanded blastocyst is, therefore, properly placed to allow an accurate shot, thanks to the possibility of movement in the x-y axis that has the microscope micromanipulation system.
- the blastocysts are fixed thanks to two clamping pipettes.
- the wavelength of the laser, as well as the number of shots necessary to isolate the MCI will be controlled by the embryologist and can vary between, some blastocysts or others.
- the trophoctoctoder cells began to expand, leaving the MCI accessible, forming a cupular structure. At this time it is when the laser is used to shoot all the trophoctoctoder cells surrounding the MCI, leaving this adjacent area free of trophoctoctoder cells and reducing the risk of dragging said cells when the MCI is under-cultivated and transferred to a new surface of fresh growth.
- DMEM Dulbecco 's Modified Eagle' s Medium
- FBS fetal bovine serum
- 0.1 M non-essential amino acids fetal bovine serum
- leukemia inhibitor factor 0.1 ⁇ -mercaptoethanol
- 50 ⁇ g / ml streptomycin 50 ⁇ g / ml streptomycin.
- Established mESCs were characterized by indirect immunohistochemistry using antibodies against SSEA-1 and Oct 3/4. Briefly, the mESC colonies were grown in special culture slots with gelatin. The cells were fixed in 4% paraformaldehyde for 20 minutes followed by 30 minutes incubation in 10% normal bovine serum in PBS. For the immunostaining of Oct 3/4, the cells are permeabilized with Triton X100 (Sigma). The colonies were incubated with the primary antibodies (1: 100 dilution in PBS) for one hour at room temperature.
- Conjugated secondary antibodies (1: 100 dilution in PBS) were used for 30 minutes at room temperature as follows: FITC-conjugated anti-mouse IgM to detect SSEA-1 and anti-mouse IgG for Oct 3/4.
- the portas were mounted in Vectashield containing DAPI.
- the primary antibodies were replaced by PBS.
- mESC Ia When it has reached confluence of the mESC Ia, these are treated with 0.05% trypsin for 5 minutes at 37 0 C, and transferred to non - adherent plates allowing Ia spontaneous differentiation and forming embryoid bodies.
- DMEM medium supplemented with 20% of
- the cells were then incubated (1 hour at room temperature) with primary antibodies to alpha-fetoprotein (Santa Cruz Biotechnology; 1: 500 dilution in PBS), anti- nestin (Chemicon; 1: 100 dilution in PBS) and antiactin (Chemicon; 1: 100 dilution in PBS).
- the slides were then incubated with a secondary antibody (biotinylate) for 30 minutes at room temperature and with a streptavidin peroxidase complex (30 minutes at room temperature). Immunostaining was visualized using diaminobenzidine and contrasted with hematoxylin. All washing steps were performed with PBS.
- the conventional karyotype analysis was determined in two different countries (p12 and p35) after having established the new mESC lines. Thirty extended metaphases per mESC line were analyzed. Two different karyotyping systems (Metasystem software or the CW4000 Karyo version 1.4) were used.
- the experimental strategy illustrated in Figure 2 was designed with the purpose of determining whether the direct culture procedure of the assisted blastocyst of the laser technique is useful for the following purposes: i) improvement of the efficiency of the isolation of the MCI and the establishment of embryonic stem cells compared with the direct culture of the blastocyst and with the laser technology separately yi ⁇ ) if it could be used successfully with low quality blastocysts, which could not have been used by laser ablation technology because the MCI would not be distinguishable for the embryologist and would normally be discarded ( Figures 1 and 2).
- the isolated MCIs were allowed to expand on MEFs or on gelatin with the double objective of: i) determining which growth surface best facilitates the establishment of embryonic stem cell lines. ii) determine the potential interference between the method of isolation of the MCI, the growth surface and the quality of the blastocysts for the derivation of embryonic stem cells.
- mouse embryonic stem cell lines were fully characterized by morphological, immunocytochemical and cytogenetic analysis, also determining the "in vitro" differentiation potential.
- the efficiency of the isolation of the MCI was not affected by the growth surface used (MEFS: 60% versus gelatin: 65% with a p ⁇ 0.05) or by the blastocyst quality (good quality: 53% versus blastocysts of poor quality 65.5%; p> 0.05) (see tables 1 and 2).
- Table 1 Efficiency of the isolation of the MCI based on the quality of the blastocyst and the method of isolation of the MCI using MEFs as a growth surface.
- MCI internal cell mass
- MEFs inactive mouse embryonic fibroblasts
- N V- Not viable Statistical differences between:
- Table 2 Efficiency of the isolation of the MCI based on the quality of the blastocyst and the method of isolation of the MCI using gelatin as a growth surface.
- the direct blastocyst culture method has several disadvantages. First, that the trophoctoctoderm cells proliferate very rapidly, repressing the growth of the MCI and second that there is a risk of overgrowth of the trophoctoctoderm and it is common for trophoctoctoder cells to be dragged when the separation of the MCI is carried out (see Figures 3A and 3B). To overcome these disadvantages, the recently developed laser ablation technique was used, based on laser shots to the trophoctoctoder cells to get rid of said unwanted cells (see Figures 3C, 3D and 3E).
- Table 3 Efficacy of the establishment of embryonic stem cells according to the quality of the blastocysts and the method of isolation of the MCI using MEFs as growth surface.
- the method of the invention provides an alternative to immunosurgery and the direct culture of the blastocyst, so that the derivation of embryonic stem cell lines can be carried out taking advantage of blastocysts (mouse and later of humans) of poor quality, fresh and especially frozen, since these constitute the main source of starting material for the derivation of embryonic stem cell lines.
- Table 4 Efficacy of the establishment of embryonic stem cells according to the quality of the blastocysts and the method of isolation of the MCI using gelatin as a growth surface.
- MCI internal cell mass
- MEFs inactive mouse embryonic fibroblasts
- mouse embryonic stem cell lines derived by means of the direct laser-assisted blastocyst culture technique show a homogeneous expression of the following markers: or alpha-fetoprotein associated with endoderm (figure 5B) or actin associated with mesoderm (figure 5C) and or nestin associated with ectoderm (figure 5D)
- the culture during prolonged periods of embryonic stem cells is associated with karyotypic changes, remaining to be resolved if the procedure of derivation of the embryonic stem cell lines is itself responsible for the loss of genetic stability. It has been analyzed if the methodology object of the present invention used for the derivation of embryonic stem cells makes embryos more vulnerable to genetic instability after prolonged culture. Using conventional karyotyping, the frequency of aneuploidy of the derived lines was evaluated using the mechanical method of direct laser culture or laser ablation.
- mice embryonic stem cell lines established by any of the methods were maintained diploid after short periods (12 passes) of culture (see table 5 and figures 6A and 6B). However, and without influence of the derivation technique used, all embryonic stem cell lines acquired an abnormal aneuploid karyotype (more than 40 chromosomes) after prolonged periods (35 passes) of culture (Table 5 and Figures 6C and 6D). These data suggest that the technique used for the derivation of embryonic stem cells does not make embryos more vulnerable to genomic instability. Table 5: Frequency of aneuploidy in mouse embryonic stem cell lines derived by direct culture of the blastocyst versus those derived by laser ablation.
- the human embryos used come from couples undergoing a cycle of fertilization "in vitró" (IVF), and have been donated to research with stem cells after the signing of an informed consent. These leftover embryos are cryopreserved in liquid nitrogen at -196 ° C.
- the success rate using this type of embryos, regarding blastocyst evolution and success in the derivation of human embryonic stem cells (hereinafter hESCs) is lower than if Fresh embryos could have been used. While fresh embryos have been able to be used in experiments with mice, Law 14/2006 on Assisted Human Reproduction Techniques allows only the use of frozen embryos left over.
- the trophoctoctoder cells began to expand, leaving the MCI accessible, forming a cupular structure. At this time it is when the laser is used to shoot all the trophoctoctoder cells surrounding the MCI, leaving this adjacent area free of trophoctoctoder cells and reducing the risk of dragging said cells when the MCI is subcultured and transferred to a New fresh growing surface. Cultivation of the primary colony
- the primary colonies of the cells obtained by the aforementioned procedure were grown on human feeders in compound medium of Dubelcco's Modified Eagle's Medium (DMEM) Knockout (80%), replacement serum (20%), non-essential amino acids (1%) , L-glutamine (1%), 2-mercaptoethanol (0.2%), at 37 ° C temperature and 5% CO 2 .
- DMEM Dubelcco's Modified Eagle's Medium
- the isolated MCIs were allowed to expand on human feeders, having previously demonstrated a better success rate on this surface, compared with the use of gelatin (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A , Barroso A, Cabrera C, Light G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by laser drilling technology enhancing the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines. "Stem. CeIIs. Develop. 2008; 17: 255-267).
- blastocysts obtained after thawing 58 embryos were classified as of good quality (27.8%), and 13 blastocysts were classified as of poor quality (72.2%). All blastocysts were released from the zona pellucida through the use of Tyrode acid, and placed individually on a layer of human feeders for the growth of the primary colony. After the relevant days of cultivation, the MCI was isolated by means of the isolation method object of the invention. Of the 5 embryos of good quality, the MCI could be isolated 4 times (80%), while with blastocysts of poor quality, 8 of 13 of blastocysts could perform this procedure (62%).
- Table 1 Efficiency of the isolation of the MCI based on the quality of the blastocyst and the method of isolation of the MCI using murine and human blastocysts.
- MCI internal cell mass
- MEFs inactive mouse embryonic fibroblasts.
- trophoctoctoderm cells proliferate very rapidly, repressing the growth of the MCI and second that there is a risk of overgrowth of the trophoctoctoderm and it is usual for the trophoctoctoder cells to be dragged when the separation of the MCI is carried out.
- mice Corntes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Light G, Montes R, Rubio R, Nieto A, Menendez P.
- the couple undergoing an IVF cycle can give a final destination to those remaining embryos that are cryopreserved in liquid nitrogen, regardless of freezing time, always with the informed consent of the couple. These embryos have been frozen for more than 5 years without the couple, or woman in their case, having claimed them.
- the different possible destinations that can be given to cryopreserved embryos are: a) Its use by the woman or spouse. b) Donation for reproductive purposes. c) Donation for research purposes. d) The cessation of its conservation without other use. Regarding the use of embryos for research purposes, it will only be authorized if it meets several requirements: a) Possession of informed consent. b) That the pre-embryo has not developed in vitro beyond 14 days after fertilization. c) That the investigation be carried out in authorized centers. d) That the investigation be carried out based on a project duly submitted and authorized by the competent authorities. e) That the possible relations of interest between centers be specified.
- blastocysts could be derived to which to submit to the process object of the invention from techniques that do not compromise the viability of the embryo, such as:
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Reproductive Health (AREA)
- Gynecology & Obstetrics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Surgery (AREA)
- Cell Biology (AREA)
- Transplantation (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pregnancy & Childbirth (AREA)
- Heart & Thoracic Surgery (AREA)
- Medical Informatics (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Method for isolation of the inner cell mass (MCI) in mammal blastocysts which includes direct slide culture of said blastocysts and the subsequent ablation by laser of the trophoectoderm of said blastocysts. The method of the invention allows for an improvement in the efficiency of the isolation of the inner cell mass of blastocysts and exploitation of low-quality blastocysts which are not suited for separation of the inner cell mass by means of the methods described in the prior art documents.
Description
TITULO TITLE
Procedimiento para el aislamiento de Ia masa celular interna en blastocistos de mamíferos.Procedure for the isolation of the internal cell mass in mammalian blastocysts.
OBJETO DE LA INVENCIÓNOBJECT OF THE INVENTION
El objeto de la presente invención es un procedimiento para el aislamiento de Ia masa celular interna (MCI) en blastocistos de mamíferos que incluye el cultivo directo en placa de dichos blastocistos y Ia posterior ablación mediante láser del trofoectodermo de dichos blastocistos. El procedimiento de Ia invención permite una mejora de Ia eficiencia del aislamiento de Ia masa celular interna de blastocistos y el aprovechamiento de blastocistos de poca calidad no aptos para Ia separación de su masa celular interna mediante los procedimientos mencionados en los documentos del estado de Ia técnica.The object of the present invention is a procedure for the isolation of the internal cell mass (MCI) in mammalian blastocysts that includes the direct plate culture of said blastocysts and the subsequent laser ablation of the trophoctoctoder of said blastocysts. The process of the invention allows an improvement of the efficiency of the isolation of the internal cell mass of blastocysts and the use of low quality blastocysts not suitable for the separation of its internal cell mass by means of the procedures mentioned in the documents of the state of the art .
ESTADO DE LA TÉCNICASTATE OF THE TECHNIQUE
En 1981 se obtuvieron por primera vez células troncales embrionarias a partir de blastocistos de ratón (Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981 ; 292:154- 56). Estas células están dotadas de dos propiedades únicas que las distinguen de todas las células troncales órgano-específicas identificadas hasta el momento:In 1981, embryonic stem cells were first obtained from mouse blastocysts (Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154-56). These cells are endowed with two unique properties that distinguish them from all organ-specific stem cells identified so far:
Primero, que se auto-renuevan continuamente en cultivo y que pueden ser conservadas y multiplicadas durante prolongados periodos de tiempo, manteniéndose en status de "no-diferenciación".First, that they continually renew themselves in cultivation and that they can be conserved and multiplied for prolonged periods of time, maintaining a status of "non-differentiation".
Segundo, que son pluripotentes, capaces de diferenciarse en cualquier tipo de célula en el cuerpo (Keller G. Embryonic stem cell differentiation: emergence of a new era ¡n biology and medicine. Genes Dev. 2005; 19:1129-55). Las primeras líneas de células troncales embrionarias humanas se derivaron en 1998 de Ia MCI de embriones no transferidos donados y congelados (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA,
Swiergiel JJ, Marshall VS, Jones JM.Embryonic stem cell lines derived from human biastocysts. Science 1998; 282:1145-7). Dichas líneas celulares mostraban las dos referidas propiedades de auto-renovación y pluripotencialidad. A partir de esa fecha, numerosos laboratorios han comunicado Ia derivación de líneas celulares troncales embrionarias humanas, a partir de embriones no transferidos, utilizando bien células alimentadoras humanas o de ratón.Second, they are pluripotent, capable of differentiating in any type of cell in the body (Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 2005; 19: 1129-55). The first human embryonic stem cell lines were derived in 1998 from the MCI of donated and frozen non-transferred embryos (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human biastocysts. Science 1998; 282: 1145-7). These cell lines showed the two referred properties of self-renewal and pluripotentiality. Since that date, numerous laboratories have reported the derivation of human embryonic stem cell lines, from non-transferred embryos, using either human or mouse feeder cells.
Debe tenerse en cuenta que Ia calidad de los blastocistos y el método de aislamiento de Ia MCI usado, son los dos factores principales que conducen al éxito o al fracaso del proceso de derivación de líneas de células troncales embrionarias humanas. Respecto a Ia calidad de los blastocistos, los humanos son de calidad más pobre que los de ratón. Ello es debido, al menos en parte, al hecho de que los blastocistos humanos están congelados cuando se intenta Ia derivación de líneas celulares troncales embrionarias humanas, debido a restricciones éticas, mientras que los blastocistos de ratón se utilizan normalmente en fresco. Ello explica no solo el extremadamente bajo grado de éxito de los procesos de derivación de líneas celulares troncales embrionarias humanas, sino que implica también el que embriones humanos de pobre calidad se descarten directamente, e incluso ni se descongelen ni se usen para este propósito.It should be taken into account that the quality of the blastocysts and the method of isolation of the MCI used, are the two main factors that lead to the success or failure of the process of derivation of human embryonic stem cell lines. Regarding the quality of blastocysts, humans are of poorer quality than mouse. This is due, at least in part, to the fact that human blastocysts are frozen when the derivation of human embryonic stem cell lines is attempted, due to ethical restrictions, while mouse blastocysts are normally used fresh. This explains not only the extremely low degree of success of the processes of derivation of human embryonic stem cell lines, but also implies that poor quality human embryos are directly discarded, and even neither defrosted nor used for this purpose.
Por otro lado, los procedimientos actuales para el aislamiento de MCI son objeto de controversia. La MCI es aislada usualmente de los blastocistos expandidos usando una amplia variedad de técnicas que incluyen Ia inmunocirugía (Solter D., Knowles BB. 1975. Immunosurgery of mouse biastocysts; Proc. Nati. Acad. Sci. U S A 72:5099-5102), procedimientos mecánicos (Bongso A, Fong CY, Ng SC, Ratnam S. 1994. Isolation and culture of inner cell mass cells from human biastocysts. Hum. Reprod. 9:2110-2117), y procedimientos de cultivo de blastocisto completo (Kim HS, Oh SK, Park YB, Anh HJ, Sung KC, Kang MJ, Lee LA, Suh CS, Kim SH, Kim DW, Moon SY. 2005. Methods for derivation of human embryonic stem cells. Stem Cells 23:1228-1233). Recientemente, se ha obtenido MCI a partir de blastómeros obtenidos de embriones en fase célula (Chung Y, Klimanskaya
I1 Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R. 2006. Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 439:216-219).On the other hand, the current procedures for the isolation of MCI are controversial. MCI is usually isolated from expanded blastocysts using a wide variety of techniques that include immunosurgery (Solter D., Knowles BB. 1975. Immunosurgery of mouse biastocysts; Proc. Nati. Acad. Sci. USA 72: 5099-5102), mechanical procedures (Bongso A, Fong CY, Ng SC, Ratnam S. 1994. Isolation and culture of inner cell mass cells from human biastocysts. Hum. Reprod. 9: 2110-2117), and complete blastocyst culture procedures (Kim HS , Oh SK, Park YB, Anh HJ, Sung KC, Kang MJ, Lee LA, Suh CS, Kim SH, Kim DW, Moon SY. 2005. Methods for derivation of human embryonic stem cells. Stem Cells 23: 1228-1233) . Recently, MCI has been obtained from blastomeres obtained from embryos in the cell phase (Chung Y, Klimanskaya I 1 Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R. 2006. Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 439: 216-219).
Todos estos procedimientos de aislamiento de MCI presentan dos inconvenientes relevantes: cambios en el cariotipo tras cultivo prolongado y Ia utilización de xeno-componentes que podrían comprometer el uso de estas líneas celulares troncales embrionarias humanas o los derivados de las mismas en potenciales aplicaciones terapéuticas. Estas células troncales embrionarias y derivados de las mismas obtenidas por los métodos indicados y cultivadas durante periodos largos, han estado asociadas con inestabilidad genética a largo plazo, aunque queda por dilucidar si ello es debido al propio proceso de derivación, o a los procesos mecánicos y/o enzimáticos utilizados para el mantenimiento rutinario de los cultivos de células troncales embrionarias. Existe claramente una demanda urgente de estudios sobre calidad de blastocistos, métodos de aislamiento de MCI y subsiguiente derivación de células troncales embrionarias, para poder dar cumplimiento a las expectativas generadas por Ia investigación con células troncales embrionarias humanas. La tecnología láser se usa habitualmente en reproducción asistida (Antinori S, Selman HA, Caff B, Panci C, Dani GL, Versad C. 1996. Zona opening of human embryos using non-contact UV láser for assisted hatching in patients with poor prognosis of pregnancy. Hum. Reprod. 11:2488-2492), porque facilita Ia transferencia nuclear, acelerando el proceso de enucleación de forma que se desarrolle en segundos, evitando al mismo tiempo cualquier daño al óvulo (Chen S., Chao K., Chang C1 Hsieh F., Ho H., Yang Y. 2004. Technical Aspects of the piezo, laser-assisted, and conventional methods for nuclear transfer of mouse oocytes and their efficiency and efficacy: Piezo minimizes damage of the ooplasmic membrane at injection. J Exp. Zool. 301 :344-351). Recientemente, se ha utilizado Ia tecnología de ablación con láser para destruir el trofoectodermo de blastocistos de ratón de buena calidad, permitiendo separar con éxito Ia MCI y Ia subsiguiente derivación de líneas
de células troncales embrionarias de ratón (Cortes JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, Concha A, Menendez P. "Evaluation of the láser technique method to isolate the inner cell mass of murine blastocysts." Biotechnol. Appl. Biochem. 2007; 46:205:209 y solicitud de patente WO03/018783) y Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Ligero G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by láser drilling technology enhances the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem. CeIIs. Dev. 2008; 17:255-267). Esta metodología ha sido recientemente demostrada en humanos (Turetsky T, Aizenman E, Gil Y1 Weinberg N, Shufaro Y, Revel A, Laufer N, Simón A, Abeliovich D, Reubinoff BE. "Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis." Hum. Reprod. 2008 ;23:46- 53) . Es importante, sin embargo, señalar que el uso de Ia tecnología de ablación con láser para destruir las células del trofoectodermo permitiendo el aislamiento de Ia MCI, requiere que se usen blastocistos expandidos de muy buena calidad con una MCI claramente distinguible, ya que si no es así, el embriólogo no puede ver con precisión y distinguir Ia MCI de las células del trofoectodermo, convirtiendo los disparos con el láser en poco precisos y aleatorios.All these MCI isolation procedures have two relevant drawbacks: changes in the karyotype after prolonged culture and the use of xeno-components that could compromise the use of these human embryonic stem cell lines or derivatives thereof in potential therapeutic applications. These embryonic stem cells and derivatives thereof obtained by the methods indicated and cultivated for long periods, have been associated with long-term genetic instability, although it remains to be elucidated if this is due to the derivation process itself, or to the mechanical processes and / or enzymatic used for routine maintenance of embryonic stem cell cultures. There is clearly an urgent demand for studies on the quality of blastocysts, methods of isolation of MCI and subsequent derivation of embryonic stem cells, in order to meet the expectations generated by research with human embryonic stem cells. Laser technology is commonly used in assisted reproduction (Antinori S, Selman HA, Caff B, Panci C, Dani GL, Versad C. 1996. Zone opening of human embryos using non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy Hum. Reprod. 11: 2488-2492), because it facilitates nuclear transfer, accelerating the enucleation process so that it develops in seconds, while avoiding any damage to the ovule (Chen S., Chao K., Chang C 1 Hsieh F., Ho H., Yang Y. 2004. Technical Aspects of the piezo, laser-assisted, and conventional methods for nuclear transfer of mouse oocytes and their efficiency and efficacy: Piezo minimizes damage of the ooplasmic membrane at injection. J Exp. Zool. 301: 344-351). Recently, laser ablation technology has been used to destroy the trophoectoderm of good quality mouse blastocysts, allowing to successfully separate the MCI and the subsequent derivation of lines of mouse embryonic stem cells (Cortes JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, Concha A, Menendez P. "Evaluation of the laser technique method to isolate the inner cell mass of murine blastocysts." Biotechnol Appl. Biochem. 2007; 46: 205: 209 and patent application WO03 / 018783) and Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Light G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by laser drilling technology enhancing the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem CeIIs. Dev. 2008; 17: 255-267). This methodology has been recently demonstrated in humans (Turetsky T, Aizenman E, Gil Y 1 Weinberg N, Shufaro Y, Revel A, Laufer N, Simon A, Abeliovich D, Reubinoff BE. "Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. "Hum. Reprod. 2008; 23: 46-53). It is important, however, to point out that the use of laser ablation technology to destroy the trophoctoctoder cells allowing the isolation of the MCI, requires that very good quality expanded blastocysts with a clearly distinguishable MCI be used, since if not Thus, the embryologist cannot see with precision and distinguish the MCI from the trophoctoctoderm cells, turning the shots with the laser into little precise and random.
El procedimiento objeto de Ia presente invención permite superar estos inconvenientes, a través de Ia combinación de una etapa de cultivo de los blastocistos completos seguida de Ia destrucción del trofoectodermo mediante ablación con láser para mejorar Ia eficacia del aislamiento de Ia MCI y de Ia derivación de células troncales embrionarias a partir de embriones de mamíferos, específicamente de ratón y humanos.The process object of the present invention allows to overcome these inconveniences, through the combination of a stage of cultivation of the complete blastocysts followed by the destruction of the trophoctoctoder by laser ablation to improve the efficiency of the isolation of the MCI and the derivation of embryonic stem cells from mammalian embryos, specifically mouse and human.
EXPLICACIÓN DE LA INVENCIÓN Constituye el objeto de Ia presente invención un procedimiento para el aislamiento de Ia MCI en blastocistos de mamíferos que incluye una etapa de ablación mediante láser del trofoectodermo de dichos blastocistos. Para
hacer más efectivo el aislamiento de Ia MCI, los blastocistos se someten, previamente a dicha etapa de ablación mediante láser, a cultivo directo en placa durante un periodo de tiempo de al menos 72 horas. En uno de los modos de realización de Ia invención, los blastocistos son de mamíferos no humanos, particularmente de ratón. En este modo de realización de Ia invención, con ratones, el cultivo directo previo a Ia ablación mediante láser incluye las siguientes sub-etapas: a) cultivo de los blastocistos recuperados de los oviductos en medio estándar, particularmente medio de cultivo G2 V.lll (Vitrolife, Sweden) a 370C y con 6% de CO2 durante un periodo de tiempo comprendido entre 24 y 48 horas. b) tratamiento de los blastocistos cultivados en Ia sub-etapa anterior para conseguir una completa eliminación de Ia zona pelúcida. c) colocación de los blastocistos tratados en Ia etapa anterior sobre una superficie de cultivo y posterior adhesión del trofoectodermo y Ia masa celular interna de los blastocistos a dicha superficie. d) mantenimiento de los blastocistos sobre dicha superficie y en un medio estándar de cultivo durante un periodo de tiempo comprendido entre 24 y 48 horas. e) refresco del medio de cultivo y mantenimiento de los blastocistos en el mismo durante un nuevo periodo de tiempo comprendido entre 24 y 48 horas.EXPLANATION OF THE INVENTION The object of the present invention constitutes a method for the isolation of MCI in mammalian blastocysts that includes a laser ablation step of the trophoctoctoder of said blastocysts. For To make the isolation of the MCI more effective, the blastocysts are subjected, previously to said laser ablation stage, to direct plate culture for a period of at least 72 hours. In one of the embodiments of the invention, the blastocysts are from non-human mammals, particularly mice. In this embodiment of the invention, with mice, the direct culture prior to laser ablation includes the following sub-stages: a) culture of the blastocysts recovered from the oviducts in standard medium, particularly G2 V.lll culture medium (Vitrolife, Sweden) at 37 0 C and with 6% CO 2 for a period of time between 24 and 48 hours. b) treatment of the blastocysts cultured in the previous sub-stage to achieve a complete elimination of the zona pellucida. c) placing the blastocysts treated in the previous stage on a culture surface and subsequent adhesion of the trophoctoctoderm and the internal cell mass of the blastocysts to said surface. d) maintenance of the blastocysts on said surface and in a standard culture medium for a period of time between 24 and 48 hours. e) refreshment of the culture medium and maintenance of the blastocysts in it for a new period of time between 24 and 48 hours.
La eliminación de Ia zona pelúcida se lleva a cabo mediante tratamiento con ácido Tyrode o enzima pronasa durante un periodo de tiempo comprendido entre 30 y 60 segundos, o bien por eclosión espontánea del blastocisto.The removal of the zona pellucida is carried out by treatment with Tyrode acid or pronase enzyme for a period of time between 30 and 60 seconds, or by spontaneous hatching of the blastocyst.
La superficie de cultivo sobre Ia cual se colocan los blastocistos tras Ia eliminación de Ia zona pelúcida es una placa recubierta con MEFs (Mouse embryonic fibroblasts) o con gelatina. El medio en el cual se mantienen los blastocistos sobre Ia superficie de cultivo es medio DMEM (Dubelcco's Modified Eagle's Médium) suplementado con suero fetal bovino al 20%, L-glutamina al 1%, aminoácidos no esenciales al 0,1 mM, 2000 lU/ml de factor inhibidor de
leucemia de ratón, β-mercaptoetanol 0,1 mM, 50 U/ml de penicilina y 50 μg/ml de estreptomicina.The culture surface on which the blastocysts are placed after the removal of the zona pellucida is a plate covered with MEFs (Mouse embryonic fibroblasts) or with gelatin. The medium in which blastocysts are maintained on the surface of the culture is DMEM (Dulbecco 's Modified Eagle Medium) supplemented with fetal bovine serum 20%, L-glutamine , 1% non - essential amino acids 0.1 mM, 2000 lU / ml inhibitory factor Mouse leukemia, 0.1 mM β-mercaptoethanol, 50 U / ml penicillin and 50 μg / ml streptomycin.
La etapa de ablación mediante láser del trofoectodermo de los blastocistos, a realizar inmediatamente tras Ia etapa de cultivo previo, se lleva a cabo con un dispositivo de diodo láser de emisión en infrarrojo a una frecuencia óptica (longitud de onda) de 1 ,48 μm., realizándose entre 20 y 100 disparos. Tras Ia ablación con láser del trofoectodermo de los blastocistos, se produce el establecimiento de las líneas celulares embrionarias de ratón a partir de Ia MCI aislada de los blastocistos sometidos al procedimiento.The laser ablation stage of the blastocyst trophoectoderm, to be performed immediately after the previous culture stage, is carried out with an infrared emission laser diode device at an optical frequency (wavelength) of 1.48 μm ., taking between 20 and 100 shots. After laser ablation of the blastocysts trophoectoderm, the establishment of mouse embryonic cell lines is produced from the MCI isolated from the blastocysts subjected to the procedure.
En otro modo de realización del procedimiento de Ia invención, los blastocistos de mamíferos son blastocistos humanos. En este caso, el cultivo directo previo a Ia ablación mediante láser incluye las siguientes sub-etapas: a) descongelación de embriones humanos en estadio de división temprana y cultivo hasta Ia evolución a blastocisto utilizando medios G1 y G2 v.5In another embodiment of the process of the invention, mammalian blastocysts are human blastocysts. In this case, the direct culture prior to laser ablation includes the following sub-stages: a) thawing of human embryos in the early division stage and culture until the blastocyst evolution using means G1 and G2 v.5
(Vitrolife, Sweden). b) tratamiento de los blastocistos cultivados en Ia sub-etapa anterior para conseguir una completa eliminación de Ia zona pelúcida. c) colocación de los blastocistos tratados en Ia etapa anterior sobre una superficie de cultivo y posterior adhesión del trofoectodermo y Ia masa celular interna de los blastocistos a dicha superficie. d) mantenimiento de los blastocistos sobre dicha superficie y en un medio estándar de cultivo durante un periodo de tiempo comprendido entre 24 y 48 horas. e) refresco del medio de cultivo y mantenimiento de los blastocistos en el mismo durante un nuevo periodo de tiempo comprendido entre 24 y 48 horas.(Vitrolife, Sweden). b) treatment of the blastocysts cultured in the previous sub-stage to achieve a complete elimination of the zona pellucida. c) placing the blastocysts treated in the previous stage on a culture surface and subsequent adhesion of the trophoctoctoderm and the internal cell mass of the blastocysts to said surface. d) maintenance of the blastocysts on said surface and in a standard culture medium for a period of time between 24 and 48 hours. e) refreshment of the culture medium and maintenance of the blastocysts in it for a new period of time between 24 and 48 hours.
La eliminación de Ia zona pelúcida se lleva acabo mediante tratamiento con ácido Tyrode o enzima pronasa durante un periodo de tiempo comprendido entre 30 y 60 segundos o bien por eclosión espontánea del blastocisto.The removal of the zona pellucida is carried out by treatment with Tyrode acid or pronase enzyme for a period of time between 30 and 60 seconds or by spontaneous hatching of the blastocyst.
La superficie. de cultivo sobre Ia cual se colocan los blastocistos tras Ia eliminación de Ia zona pelúcida es una placa recubierta con "feedérs"
humanos y el medio en el cual se mantienen los blastocistos sobre Ia superficie de cultivo es medio DMEM (Dubelcco's Modified Eagle's Médium) Knockout (80%), suero de reemplazo (20%), aminoácidos no esenciales (1%), L-glutamina (1%), 2-mercaptoetanol (0,2%), a 37° C y 5% de CO2. La etapa de ablación mediante láser del trofoectodermo de los blastocistos, a realizar inmediatamente tras Ia etapa de cultivo previo, se lleva a cabo con un dispositivo de diodo láser de emisión en infrarrojo a una frecuencia óptica (longitud de onda) de 1 ,48 μm, realizándose entre 20 y 100 disparos. Tras Ia ablación con láser del trofoectodermo de los blastocistos, se produce el establecimiento de las líneas celulares embrionarias humanas a partir de Ia MCI aislada de los blastocistos sometidos al procedimiento.The surface. of culture on which the blastocysts are placed after the removal of the zona pellucida is a plate covered with "feedérs" human and the environment in which blastocysts on the surface culture are maintained is DMEM (Dulbecco 's Modified Eagle Medium) Knockout (80%), serum replacement (20%), non - essential (1%) amino acids, L -glutamine (1%), 2-mercaptoethanol (0.2%), at 37 ° C and 5% CO 2 . The laser ablation stage of the blastocyst trophoectoderm, to be performed immediately after the previous culture stage, is carried out with an infrared emission laser diode device at an optical frequency (wavelength) of 1.48 μm , taking between 20 and 100 shots. After the laser ablation of the blastocysts trophoectoderm, the establishment of human embryonic cell lines from the MCI isolated from the blastocysts subjected to the procedure occurs.
BREVE DESCRIPCIÓN DE LAS FIGURASBRIEF DESCRIPTION OF THE FIGURES
Figura 1 : Figuras representativas de Ia clasificación de calidad de los blastocistos de ratón obtenidos.Figure 1: Representative figures of the quality classification of the blastocysts obtained.
A. Embrión en estadio de blastocisto compacto.A. Embryo in compact blastocyst stage.
B. Embrión en estadio de blastocisto expandido.B. Embryo in expanded blastocyst stage.
C. Blastocisto de alto crecimiento y buena calidad con una masa celular interna (MCI) grande y claramente distinguible. D. Blastocistos de bajo crecimiento con una masa celular interna pequeña claramente distinguible.C. High-growth and good-quality blastocyst with a large and clearly distinguishable internal cell mass (MCI). D. Low growth blastocysts with a clearly distinguishable small internal cell mass.
E. Blastocistos de bajo crecimiento con masa celular interna no distinguible. La flecha negra indica dónde se localiza Ia región de Ia MCI.E. Low growth blastocysts with internal cell mass not distinguishable. The black arrow indicates where the region of the MCI is located.
Figura 2: Diseño experimental para Ia optimización de Ia eficiencia de Ia derivación de células troncales embrionarias de ratón basada en Ia influencia de Ia calidad del blastocisto, método de aislamiento de Ia masa celular interna y superficie de crecimiento.
Figura 3: Gráficas representativas de Ia valoración de los distintos métodos de aislamiento de Ia masa celular interna en blastocistos de ratón.Figure 2: Experimental design for the optimization of the efficiency of the derivation of mouse embryonic stem cells based on the influence of the quality of the blastocyst, method of isolation of the internal cell mass and growth surface. Figure 3: Graphs representative of the assessment of the different methods of isolation of the internal cell mass in mouse blastocysts.
(A-B) Aislamiento de Ia MCI por el método de cultivo de blastocisto completo.(A-B) Isolation of the MCI by the complete blastocyst culture method.
A) Crecimiento de un blastocisto adherido a MEFs a los cinco días del cultivo del blastocisto completo.A) Growth of a blastocyst attached to MEFs five days after the cultivation of the complete blastocyst.
B) Crecimiento de un blastocisto adherido a gelatina a los cinco días del cultivo del blastocisto completo. (C-E) Aislamiento de Ia MCI mediante Ia tecnología de ablación con láser.B) Growth of a blastocyst attached to gelatin five days after the cultivation of the complete blastocyst. (C-E) Isolation of the MCI by means of laser ablation technology.
C) Blastocisto fijado por dos pipetas con liberación de Ia MCI mediante ablación con láser del trofoectodermo y de Ia zona pelúcida. Las puntas de flecha negras representan los disparos de láser.C) Blastocyst fixed by two pipettes with release of the MCI by laser ablation of the trophoctoctoderm and the pellucid zone. The black arrowheads represent the laser shots.
D) El blastocisto de C) justo tras Ia ruptura del trofoectodermo por el disparo del láser. La flecha negra muestra el trofoectodermo lisado por el láser.D) The blastocyst of C) just after the rupture of the trophoctoctoder by the firing of the laser. The black arrow shows the trophoectoderm lysed by the laser.
E) Crecimiento del mismo blastocisto dos días después de Ia ablación directa con láser.E) Growth of the same blastocyst two days after direct laser ablation.
(F-G) Aislamiento de Ia MCI mediante cultivo previo del blastocisto completo y subsiguiente ablación con láser del trofoectodermo.(F-G) Isolation of the MCI by previous culture of the complete blastocyst and subsequent laser ablation of the trophoctoctoderm.
F) Crecimiento del blastocisto a los 5 días del cultivo del blastocisto completo.F) Growth of the blastocyst 5 days after the culture of the complete blastocyst.
G) MCI limpia y libre de células de trofoectodermo tras los disparos de láser. H) Trofoectodermo residual tras Ia ablación con láser. Los asteriscos representan las MCIs. Las flechas blancas anchas indican Ia zona pelúcida del embrión. Las flechas blancas finas indican Ia situación exacta de los disparos de láser.G) Clean and free MCI of trophoctoctore cells after laser shots. H) Residual trophoectoderm after laser ablation. Asterisks represent MCIs. The wide white arrows indicate the hairy area of the embryo. The fine white arrows indicate the exact situation of the laser shots.
Figura 4: Gráficas representativas que muestran Ia derivación conseguida y Ia caracterización inmunocitoquímica de las células troncales embrionarias de ratón,
A) Morfología típica (magnificación 10 X) de líneas de células troncales embrionarias sobre MEFs establecidas mediante cultivo previo del blastocisto completo y posterior ablación con láser del trofoectodermo.Figure 4: Representative graphs showing the derivation achieved and the immunocytochemical characterization of mouse embryonic stem cells, A) Typical morphology (10 X magnification) of embryonic stem cell lines on MEFs established by prior culture of the complete blastocyst and subsequent laser ablation of the trophoctoctoderm.
B) Magnificación 40 X de Ia misma colonia de células troncales embrionarias.B) 40 X magnification of the same colony of embryonic stem cells.
C) Expresión del SSEA-1 (verde). Contraste del núcleo con DAPI (azul).C) Expression of SSEA-1 (green). Contrast of the nucleus with DAPI (blue).
D) Expresión de Oct3/4 (rojo).D) Expression of Oct3 / 4 (red).
Figura 5: Mantenimiento del potencial de diferenciación de las tres capas germinales de las líneas de células troncales embrionarias de ratón.Figure 5: Maintenance of the differentiation potential of the three germ layers of mouse embryonic stem cell lines.
A) Representación de blastocistos expandidos.A) Representation of expanded blastocysts.
B) Expresión de alfafetoproteína (endodermo).B) Expression of alphafetoprotein (endoderm).
C) Expresión de actina (mesodermo). D) Expresión de nestina (ectodermo).C) Actin expression (mesoderm). D) Expression of nestin (ectoderm).
Figura 6: Caracterización citogenética de las líneas establecidas de células troncales embrionarias de ratón.Figure 6: Cytogenetic characterization of established mouse embryonic stem cell lines.
A) Cariotipo diploide representativo de una línea de células troncales embrionarias establecida mediante cultivo del blastocisto completo trasA) Representative diploid karyotype of an embryonic stem cell line established by complete blastocyst culture after
12 pases en cultivo in vitro.12 passes in vitro culture.
B) Cariotipo diploide representativo de una línea de células troncales embrionarias establecida mediante tecnología de ablación con láser tras 12 pases en cultivo in vitro. C) Cariotipo aneuploide representativo de una línea de células troncales embrionarias establecida mediante cultivo del blastocisto completo tras cultivo extensivo in vitro.B) Representative diploid karyotype of an embryonic stem cell line established by laser ablation technology after 12 passes in vitro culture. C) Representative aneuploid karyotype of an embryonic stem cell line established by culturing the complete blastocyst after extensive in vitro culture.
D) Cariotipo aneuploide representativo de una línea de células troncales embrionarias establecida mediante tecnología de ablación con láser tras cultivo extensivo in vitro.
Figura 7: Figuras representativas de Ia clasificación de calidad de los embriones y blastocistos humanos obtenidos.D) Aneuploid karyotype representative of an embryonic stem cell line established by laser ablation technology after extensive in vitro culture. Figure 7: Representative figures of the quality classification of the human embryos and blastocysts obtained.
A. Embrión en estadio de división temprana de buena calidad inmediatamente después de Ia descongelación. B. Embrión en estadio de división temprana de mala calidad inmediatamente después de Ia descongelación. . .A. Embryo in early division stage of good quality immediately after thawing. B. Embryo in an early stage of poor quality immediately after thawing. . .
C. Blastocisto de alto crecimiento y buena calidad con una masa celular interna (MCI) grande y claramente distinguible. La flecha negra indica dónde se localiza Ia región de Ia MCI. D. Blastocisto de bajo crecimiento con masa celular interna no distinguible.C. High-growth and good-quality blastocyst with a large and clearly distinguishable internal cell mass (MCI). The black arrow indicates where the region of the MCI is located. D. Low growth blastocyst with internal cell mass not distinguishable.
Figura 8: Diseño experimental para Ia optimización de Ia eficiencia de Ia derivación de células troncales embrionarias humanas basada en Ia influencia de Ia calidad del blastocisto, superficie de crecimiento y método de aislamiento de Ia masa celular interna.Figure 8: Experimental design for the optimization of the efficiency of the derivation of human embryonic stem cells based on the influence of the quality of the blastocyst, growth surface and method of isolation of the internal cell mass.
Figura 9: Imágenes representativas de Ia evolución a blastocisto de un preembrión humano descongelado y valoración del método de aislamiento de Ia masa celular interna mediante cultivo directo previo a ablación con láser.Figure 9: Representative images of the blastocyst evolution of a thawed human pre-embryo and assessment of the method of isolation of the internal cell mass by direct culture prior to laser ablation.
(A-C) Evolución de un preembrión en estadio de división temprana hasta el estadio de blastocisto.(A-C) Evolution of a pre-embryo in the early division stage to the blastocyst stage.
A) Preembrión en día +3 de desarrollo, inmediatamente después de ser descongelado. B) Preembrión en día +4 de desarrollo, donde observamos reactivación del proceso de división celular.A) Pre-embryo on day +3 of development, immediately after being thawed. B) Pre-embryo on day +4 of development, where we observe reactivation of the cell division process.
C) Preembrión en día +6. de desarrollo, donde ha alcanzado el estadio de blastocisto expandido.
(D-F) Aislamiento de Ia MCI mediante cultivo previo del blastocisto completo y subsiguiente ablación con láser del trofoectodermo.C) Pre-embryo in day +6. of development, where it has reached the stage of expanded blastocyst. (DF) Isolation of the MCI by previous culture of the complete blastocyst and subsequent laser ablation of the trophoctoctoderm.
D) Crecimiento del blastocisto a los 5 días del cultivo del blastocisto ' completo. E) Crecimiento del blastocisto a los 5 días del cultivo del blastocisto completo una vez sometido a Ia ablación con láser. Las flechas blancas anchas indican Ia zona pelúcida del embrión. Las flechas blancas finas indican Ia situación exacta de los disparos de láser.D) growth at 5 days blastocyst cultivation 'full blastocyst. E) Growth of the blastocyst 5 days after the culture of the complete blastocyst once it was subjected to laser ablation. The wide white arrows indicate the hairy area of the embryo. The fine white arrows indicate the exact situation of the laser shots.
F) MCI limpia y libre de células de trofoectodermo tras los disparos de láser.F) Clean and free MCI of trophoectoderm cells after laser shots.
DESCRIPCIÓN DETALLADA Y MODO DE REALIZACIÓN DE LADETAILED DESCRIPTION AND MODE OF REALIZATION OF THE
INVENCIÓNINVENTION
A) Blastocistos de ratón Obtención de blastocistos de ratón y evaluación de Ia calidad blastocitariaA) Mouse blastocysts Obtaining mouse blastocysts and evaluation of blastocyte quality
Los blastocistos fueron obtenidos mediante el lavado de los oviductos de ratonas de Ia cepa C57BL/CBA de 3 a 6 meses de edad. Estas ratonas habían sido sacrificadas previamente por dislocación cervical en el día 4,5 de embarazo (contando que el periodo de copulación ocurre en día 0.5). Los oviductos fueron diseccionados y colocados en medio PBS atemperado a 370C y posteriormente fueron cuidadosamente manipulados en una placa de Petri con .medio atemperado G-MOPS (Vitrolife, Sweden) como se describe en Tanaka N. et al. "Laser-assisted blastocyst dissection and subsequent ; cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model" J. Transí. Med. 2006; 4:20-32.The blastocysts were obtained by washing the oviducts of mice of the C57BL / CBA strain from 3 to 6 months of age. These mice had been previously sacrificed by cervical dislocation on day 4.5 of pregnancy (counting that the coupling period occurs on day 0.5). The oviducts were dissected and placed in PBS medium tempered at 37 0 C and were subsequently carefully handled in a Petri dish with .medio tempered G-MOPS (Vitrolife, Sweden) as described in Tanaka N. et al. "Laser-assisted blastocyst dissection and subsequent; cultivation of embryonic stem cells in a serum / cell free culture system: applications and preliminary results in a murine model" J. Transí. Med. 2006; 4: 20-32.
Los oviductos fueron separados de Ia porción del útero y de los ovarios mediante sección con bisturí. Este procedimiento fue realizado con Ia ayuda
de una lupa estereoscópica y un microscopio invertido con óptica Hoffmán. Los blastocistos recuperados fueron colocados en medio de cultivo G2 V.lll (Vitrolife, Sweden) en un incubador a 370C y 6% de CO2 como se ha descrito en Ia referencia mencionada de Tanaka et al. . Ciento once blastocistos de ratón fueron incluidos en el presente estudio que presta soporte experimental al procedimiento de Ia invención." Fueron recuperados en estadio de compactación o en estadio de blastocisto expandido. Los blastocistos en estadio de compactación fueron cultivados hasta el estadio de blastocisto expandido. Con el objetivo de identificar el método más eficaz.: respecto al aislamiento de Ia MCI y el posterior establecimiento de . las líneas, de células troncales embrionarias de ratón (mESC), se tuvo en cuenta Ia calidad blastocitaria. Debido a Ia similitud en Ia morfología entre los blastocistos humanos y de ratón, se adoptó el mismo sistema de calidad blastocitaria humana utilizado por Ia Universidad de Cornell (Ithaca, NY). En el proceso de derivación, los blastocistos fueron clasificados como blastocistos de buena o de mala calidad: los blastocistos de buena calidad poseían una MCI grande y claramente distinguible. Los blastocistos de mala calidad fueron clasificados cuando poseían una MCI pequeña pero distinguible y cuando poseían una MCI indistinguible.The oviducts were separated from the portion of the uterus and the ovaries by means of a scalpel section. This procedure was performed with the help of a stereoscopic magnifying glass and an inverted microscope with Hoffman optics. The recovered blastocysts were placed in culture medium G2 V.lll (Vitrolife, Sweden) in an incubator at 37 0 C and 6% CO2 as described in the above reference of Tanaka et al. . One hundred and eleven mouse blastocysts were included in the present study that provides experimental support to the method of the invention. " They were recovered in the compaction stage or in the expanded blastocyst stage. The blastocysts in the compaction stage were grown to the expanded blastocyst stage. In order to identify the most effective method: with respect to the isolation of the MCI and subsequent establishment of the lines, of mouse embryonic stem cells (mESC), the blastocyte quality was taken into account Due to the similarity in the morphology between the human and mouse blastocysts, the same human blastocyte quality system used by Ia was adopted. Cornell University (Ithaca, NY) In the derivation process, blastocysts were classified as blastocysts of good or poor quality: blastocysts of good quality possessed a large and clearly distinguishable MCI. Blastocysts of poor quality were classified when they possessed a small but distinguishable MCI and when they had an indistinguishable MCI.
Métodos de aislamiento de Ia MCI en blastocistos de ratón. Se utilizaron tres métodos experimentales diferentes para realizar el aislamiento de Ia MCI en blastocistos de ratón y el establecimiento de mESC. La figura 2 muestra el diseño experimental realizado para aumentar Ia eficiencia del. aislamiento de Ia MCI en blastocistos de ratón y el establecimiento de células troncales embrionarias de ratón sobre Ia base de. Ia calidad blastocitaria, el método de aislamiento y Ia superficie de crecimiento utilizada.Methods of isolation of MCI in mouse blastocysts. Three different experimental methods were used to perform the isolation of the MCI in mouse blastocysts and the establishment of mESC. Figure 2 shows the experimental design performed to increase the efficiency of the. isolation of the MCI in mouse blastocysts and the establishment of mouse embryonic stem cells on the basis of. The blastocyte quality, the method of isolation and the growth surface used.
Método de cultivo directo del blastocistoDirect blastocyst culture method
El blastocisto fue cultivado directamente de tal manera que tanto las células de Ja MCI como las del trofoectodermo se adhieren a los MEFs o a Ia
gelatina. Dos días después,, se confirmó Ia adhesión del blastocisto a Ia superficie de crecimiento y el medio de cultivo fue cambiado por medio fresco atemperado. En el día 3 de cultivo las células comenzaron a expandirse proporcionando un apoyo al crecimiento de Ia MCI creciendo y formando una estructura en forma de cúpula. En este momento, la MCI fue cuidadosamente levantada y transferida a una superficie de crecimiento fresca.The blastocyst was directly cultured in such a way that both Ja MCI and trophoctoctoder cells adhere to MEFs or Ia jelly. Two days later, the blastocyst adhesion to the growth surface was confirmed and the culture medium was changed to fresh tempered medium. On day 3 of culture the cells began to expand providing support for the growth of the MCI by growing and forming a dome-shaped structure. At this time, the MCI was carefully raised and transferred to a fresh growing surface.
El método de cultivo directo tiene el riesgo de sobrecrecimiento del trofoectodermo, ya que este es cultivado en su totalidad junto con Ia MCI, dificultando al embriólogo el acceso a Ia MCI en blastocistos de mala calidad.The direct culture method has the risk of overgrowth of the trophoctoctoder, since it is fully cultivated together with the MCI, making it difficult for the embryologist to access the MCI in blastocysts of poor quality.
Tecnología láserLaser technology
El sistema láser utilizado para el procedimiento de Ia invención (OCTAX EyewareTM, Alemania) está conectado a un microscopio invertido (IX-71 , Olympus) y procesado mediante un programa informático el cual permite analizar los datos. El uso del láser consiste en disparar y lisar las células del trofoectodermo buscando su separación. Esta técnica de disparo directo sólo puede ser usada en blastocistos de ratón de buena calidad, donde Ia MCI es claramente, identificable y distinguible de las células del trofoectodermo. El láser es colocado en una zona determinada del campo localizado en Ia pantalla de ordenadora modo de diana. El blastocisto expandido es, por tanto, colocado adecuadamente para permitir un disparo certero, gracias a Ia posibilidad dé movimiento en el eje x-y que posee el sistema de micromanipulación del microscopio. Para ello los blastocistos son fijados gracias a dos pipetas de sujeción. La longitud de onda del láser, así como el número de disparos necesarios para aislar la MCI será controlado por el embriólogo y puede variar entre, unos blastocistos u otros.The laser system used for the process of the invention (OCTAX EyewareTM, Germany) is connected to an inverted microscope (IX-71, Olympus) and processed by a computer program which allows data to be analyzed. The use of the laser consists of firing and lysing the trophoctoctoder cells looking for their separation. This direct firing technique can only be used in mouse blastocysts of good quality, where the MCI is clearly, identifiable and distinguishable from trophoectoderm cells. The laser is placed in a certain area of the field located in the target mode sorting screen. The expanded blastocyst is, therefore, properly placed to allow an accurate shot, thanks to the possibility of movement in the x-y axis that has the microscope micromanipulation system. For this, the blastocysts are fixed thanks to two clamping pipettes. The wavelength of the laser, as well as the number of shots necessary to isolate the MCI will be controlled by the embryologist and can vary between, some blastocysts or others.
Cultivo directo del blastocisto ayudado de Ia técnica láserDirect culture of the blastocyst aided by the laser technique
Es el objetivó principal de Ia presente invención. En un intento de mejorar Ia. metodología en el proceso de aislamiento de Ia MCI y posterior
establecimiento de mESC a partir de blastocistos expandidos de mala calidad, se ha desarrollado un nuevo método, consistente en dos pasos, i) cultivo directo del blastocisto y, ii) tecnología láser. Con este método, todos los blastocistos de ratón fueron tratados con ácido Tyrode para Ia completa disolución de Ia zona pelúcida durante no más de un minuto. Los blastocistos fueron colocados en Ia superficie de crecimiento buscando Ia adhesión de Ia MCI y las células del trofoectodermo. Dos días después, dicha adhesión fue confirmada y se procedió al cambio de medio por medio nuevo y fresco. En torno al día 3 de cultivo, las células del trofoectodermo comenzaron a expandirse, dejando Ia MCI accesible, formando una estructura cupular. En este momento es cuando se utiliza el láser para disparar a todas las células del trofoectodermo que rodean a Ia MCI, dejando esta área adyacente libre de células del trofoectodermo y reduciendo el riesgo de arrastre de dichas células cuando Ia MCI sea sub-cultivada y transferida a una nueva superficie de crecimiento fresca.It is the main objective of the present invention. In an attempt to improve Ia. methodology in the process of isolation of the MCI and later establishment of mESC from expanded blastocysts of poor quality, a new method has been developed, consisting of two steps, i) direct culture of the blastocyst and, ii) laser technology. With this method, all mouse blastocysts were treated with Tyrode acid for the complete dissolution of the zona pellucida for no more than one minute. The blastocysts were placed on the growth surface seeking the adhesion of the MCI and the trophoctoctoder cells. Two days later, said adhesion was confirmed and the medium was changed by fresh and new medium. Around day 3 of culture, the trophoctoctoder cells began to expand, leaving the MCI accessible, forming a cupular structure. At this time it is when the laser is used to shoot all the trophoctoctoder cells surrounding the MCI, leaving this adjacent area free of trophoctoctoder cells and reducing the risk of dragging said cells when the MCI is under-cultivated and transferred to a new surface of fresh growth.
Cultivo de Ia colonia primaríaCultivation of the primary colony
Las colonias primarias de las células obtenidas por los procedimientos mencionados fueron cultivadas sobre MEFs o placas con gelatina en medio compuesto de Dubelcco's Modified Eagle's Médium (DMEM) suplementado con 20% de suero fetal bovino (FBS), 1% L-glutamina, 0,1 M de aminoácidos no esenciales, factor inhibidor de Ia leucemia, 0,1 β-mercaptoetanol, y 50 μg/ml de estreptomicina.Primary colonies of the cells obtained by the methods mentioned were cultured on MEFs or plates with gelatin in medium composed of Dulbecco 's Modified Eagle' s Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 1% L- glutamine, 0.1 M non-essential amino acids, leukemia inhibitor factor, 0.1 β-mercaptoethanol, and 50 μg / ml streptomycin.
Superficies de crecimientoGrowth surfaces
Para determinar que superficie de crecimiento facilita el aislamiento de Ia MCI y el establecimiento de las mESC, las colonias primarias fueron cultivadas individualmente sobre MEFs o sobre placas con gelatina, como se describió previamente (Cortes JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, Concha A, Menéndez P. "Evaluation of the láser technique method to isolate the inner cell mass of murine blastocysts"; Biotechnol. Appl. Biochem. 2007; 46:205-209)
Caracterización inmunohistoquímica de las mESC establecidasTo determine which growth surface facilitates the isolation of the MCI and the establishment of the mESC, the primary colonies were grown individually on MEFs or on plates with gelatin, as previously described (Cortes JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, Concha A, Menéndez P. "Evaluation of the laser technique method to isolate the inner cell mass of murine blastocysts"; Biotechnol. Appl. Biochem. 2007; 46: 205-209) Immunohistochemical characterization of established mESC
Las mESC establecidas fueron caracterizadas por inmunohistoquímica indirecta usando anticuerpos contra el SSEA-1 y el Oct 3/4. Brevemente, las colonias de mESC fueron cultivadas en portas de cultivo especiales con gelatina. Las células fueron fijadas en 4% de paraformaldehido durante 20 minutos seguidos de 30 minutos de incubación en 10% de suero normal bovino en PBS. Para Ia inmunotinción de Oct 3/4, las células son permeabilizadas con Tritón X100 (Sigma). Las colonias fueron incubadas con los anticuerpos primarios (dilución 1 :100 en PBS) durante una hora a temperatura ambiente. Los anticuerpos secundarios conjugados (dilución 1 :100 en PBS) fueron usados durante 30 minutos a temperatura ambiente como sigue: FITC-conjugated anti-mouse IgM para detectar SSEA-1 y anti- mouse IgG para Oct 3/4. Los portas fueron montados en Vectashield conteniendo DAPI. Para el control negativo de tinción los anticuerpos primarios fueron reemplazados por PBS.Established mESCs were characterized by indirect immunohistochemistry using antibodies against SSEA-1 and Oct 3/4. Briefly, the mESC colonies were grown in special culture slots with gelatin. The cells were fixed in 4% paraformaldehyde for 20 minutes followed by 30 minutes incubation in 10% normal bovine serum in PBS. For the immunostaining of Oct 3/4, the cells are permeabilized with Triton X100 (Sigma). The colonies were incubated with the primary antibodies (1: 100 dilution in PBS) for one hour at room temperature. Conjugated secondary antibodies (1: 100 dilution in PBS) were used for 30 minutes at room temperature as follows: FITC-conjugated anti-mouse IgM to detect SSEA-1 and anti-mouse IgG for Oct 3/4. The portas were mounted in Vectashield containing DAPI. For the negative control of staining the primary antibodies were replaced by PBS.
Formación de cuerpos embrioides y análisis de Ia diferenciaciónEmbryoid body formation and differentiation analysis
Cuando se ha alcanzado Ia confluencia de las mESC, estas son tratadas con 0,05% de tripsina durante 5 minutos a 370C, y son transferidas a placas no- adherentes permitiendo Ia diferenciación espontánea y formando los cuerpos embrioides. Para ello se utiliza medio DMEM suplementado con 20% deWhen it has reached confluence of the mESC Ia, these are treated with 0.05% trypsin for 5 minutes at 37 0 C, and transferred to non - adherent plates allowing Ia spontaneous differentiation and forming embryoid bodies. For this, DMEM medium supplemented with 20% of
FBS, 1% de L-glutamina, 0,1 mM de aminoácidos no-esenciales y 0,1 mM de β-mercaptoetanol, pero sin añadir LIF. Los cambios de medio se realizan cada 2-3 días. Transcurridos 21 días de Ia diferenciación de los cuerpos embrioides, estos fueron trasferidos a una placa tratada donde crecieron y dieron lugar a un cultivo confluente en mono-capa. La capacidad de diferenciación a las tres capas germinales fue evaluada por inmunohistoquímica. Las células de los cuerpos embrioides fueron fijadas con formaldehído durante 10 minutos. A continuación, las células fueron incubadas (1 hora a temperatura ambiente) con anticuerpos primarios para alfa-fetoproteina (Santa Cruz Biotechnology; dilución 1 :500 en PBS), anti-
nestina (Chemicon; dilución 1 :100 en PBS) y antiactina (Chemicon; dilución 1:100 en PBS). Los portas fueron entonces incubados con un anticuerpo secundario (biotinilato) durante 30 minutos a temperatura ambiente y con un complejo de estreptavidina peroxidasa (30 minutos a temperatura ambiente). La inmunotinción fue visualizada usando diaminobencidina y contrastado con hematoxilina. Todos los pasos de lavado fueron realizados con PBS.FBS, 1% L-glutamine, 0.1 mM non-essential amino acids and 0.1 mM β-mercaptoethanol, but without adding LIF. Media changes are made every 2-3 days. After 21 days of the differentiation of the embryoid bodies, these were transferred to a treated plate where they grew and gave rise to a confluent mono-layer culture. The differentiation capacity to the three germ layers was evaluated by immunohistochemistry. Embryoid body cells were fixed with formaldehyde for 10 minutes. The cells were then incubated (1 hour at room temperature) with primary antibodies to alpha-fetoprotein (Santa Cruz Biotechnology; 1: 500 dilution in PBS), anti- nestin (Chemicon; 1: 100 dilution in PBS) and antiactin (Chemicon; 1: 100 dilution in PBS). The slides were then incubated with a secondary antibody (biotinylate) for 30 minutes at room temperature and with a streptavidin peroxidase complex (30 minutes at room temperature). Immunostaining was visualized using diaminobenzidine and contrasted with hematoxylin. All washing steps were performed with PBS.
Análisis citogenéticoCytogenetic analysis
El análisis del cariotipo convencional fue determinado en dos pases diferentes (p12 y p35) después de haber establecido las nuevas líneas mESC. Treinta metafases extendidas por línea de mESC fueron analizadas. Se utilizaron dos sistemas de cariotipación diferentes (Metasystem software o el CW4000 Karyo versión 1.4).The conventional karyotype analysis was determined in two different countries (p12 and p35) after having established the new mESC lines. Thirty extended metaphases per mESC line were analyzed. Two different karyotyping systems (Metasystem software or the CW4000 Karyo version 1.4) were used.
Análisis estadísticoStatistic analysis
Para estimar si las diferencias encontradas eran significativas entre los distintos métodos usados, se realizó el test de Ia chi-cuadrado de Pearson y Ia correlación de Yate. Todos los análisis fueron realizados con el programa Statgrafic (versión 5.0). Se consideraron diferencias significativas cuando el valor de p fue menor de 0,05.To estimate if the differences found were significant between the different methods used, the Pearson's chi-square test and the Yate correlation were performed. All analyzes were performed with the Statgrafic program (version 5.0). Significant differences were considered when the p value was less than 0.05.
Resultados obtenidosResults obtained
De los 111 blastocistos de ratón, 88 de ellos (el 79%) eran blastocistos expandidos de buena calidad y 23 de los 111 (el 21%), eran blastocistos expandidos de mala calidad (ver Figura 1).Of the 111 mouse blastocysts, 88 of them (79%) were expanded blastocysts of good quality and 23 of the 111 (21%) were expanded blastocysts of poor quality (see Figure 1).
Sobre Ia base de esta distribución de Ia calidad de los blastocistos, se diseñó Ia estrategia experimental ilustrada en Ia Figura 2 con el propósito de determinar si el procedimiento de cultivo directo del blastocisto ayudado de Ia técnica láser es útil para los siguientes propósitos: i) mejora de Ia eficiencia del aislamiento de Ia MCI y del establecimiento de células troncales embrionarias comparado
con el cultivo directo del blastocisto y con Ia tecnología láser por separado y i¡) si se podría utilizar con éxito con blastocistos de baja calidad, los cuales no podrían haberse usado mediante Ia tecnología de ablación con láser debido a que Ia MCI no sería distinguible para el embriólogo y serían normalmente descartados (Figuras 1 y 2).On the basis of this distribution of the quality of the blastocysts, the experimental strategy illustrated in Figure 2 was designed with the purpose of determining whether the direct culture procedure of the assisted blastocyst of the laser technique is useful for the following purposes: i) improvement of the efficiency of the isolation of the MCI and the establishment of embryonic stem cells compared with the direct culture of the blastocyst and with the laser technology separately yi¡) if it could be used successfully with low quality blastocysts, which could not have been used by laser ablation technology because the MCI would not be distinguishable for the embryologist and would normally be discarded (Figures 1 and 2).
Las MCI aisladas se dejaron expandir sobre MEFs o sobre gelatina con el doble objetivo de: i) determinar que superficie de crecimiento facilita mejor el establecimiento de líneas de células troncales embrionarias. ii) determinar Ia interferencia potencial entre el método de aislamiento de Ia MCI, Ia superficie de crecimiento y Ia calidad de los blastocistos para la derivación de células troncales embrionarias.The isolated MCIs were allowed to expand on MEFs or on gelatin with the double objective of: i) determining which growth surface best facilitates the establishment of embryonic stem cell lines. ii) determine the potential interference between the method of isolation of the MCI, the growth surface and the quality of the blastocysts for the derivation of embryonic stem cells.
Finalmente, las líneas de células troncales embrionarias de ratón se caracterizaron completamente mediante análisis morfológico, inmunocitoquímico y citogenético, determinándose también el potencial de diferenciación "in vitro".Finally, mouse embryonic stem cell lines were fully characterized by morphological, immunocytochemical and cytogenetic analysis, also determining the "in vitro" differentiation potential.
Mejora de Ia eficiencia del aislamiento de Ia MCI mediante cultivo directo del blastocisto ayudado de Ia técnica láser sin importar la calidad del blastocisto y la superficie de crecimiento.Improvement of the efficiency of the isolation of the MCI by direct culture of the blastocyst aided by the laser technique regardless of the quality of the blastocyst and the growth surface.
Mediante el procedimiento de cultivo directo del blastocisto, Ia eficiencia del aislamiento de la MCI no se vio afectada ni por Ia superficie de crecimiento utilizada (MEFS: 60% frente a gelatina: 65% con una p≥0,05) ni por, Ia calidad del blastocisto (buena calidad: 53% frente a blastocistos de mala calidad 65,5%; p>0,05) (ver tablas 1 y 2).
Tabla 1: Eficiencia del aislamiento de Ia MCI basado en Ia calidad del blastocisto y en el método de aislamiento de Ia MCI usando MEFs como superficie de crecimiento.By means of the direct culture of the blastocyst, the efficiency of the isolation of the MCI was not affected by the growth surface used (MEFS: 60% versus gelatin: 65% with a p≥0.05) or by the blastocyst quality (good quality: 53% versus blastocysts of poor quality 65.5%; p> 0.05) (see tables 1 and 2). Table 1: Efficiency of the isolation of the MCI based on the quality of the blastocyst and the method of isolation of the MCI using MEFs as a growth surface.
Acrónimos: MCI.- masa celular interna; MEFs.- fibroblastos embrionarios de ratón inactivos; N. V- No viable Diferencias estadísticas entre:Acronyms: MCI.- internal cell mass; MEFs.- inactive mouse embryonic fibroblasts; N. V- Not viable Statistical differences between:
1 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a cultivo directo del blastocisto para blastocistos de buena calidad: p=0.005. 1 Direct culture of the blastocyst aided by the laser technique versus direct culture of the blastocyst for blastocysts of good quality: p = 0.005.
2 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a ablación con láser para blastocistos de buena calidad: p=0.001. 2 Direct culture of the blastocyst aided by the laser technique against laser ablation for blastocysts of good quality: p = 0.001.
3 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a cultivo directo del blastocisto para blastocistos de mala calidad: p=0.05.
3 Direct culture of the blastocyst aided by the laser technique versus direct culture of the blastocyst for blastocysts of poor quality: p = 0.05.
Tabla 2: Eficiencia del aislamiento de Ia MCI basado en Ia calidad del blastocisto y en el método de aislamiento de Ia MCI usando gelatina como superficie de crecimiento.Table 2: Efficiency of the isolation of the MCI based on the quality of the blastocyst and the method of isolation of the MCI using gelatin as a growth surface.
Acrónimos: MCI.- masa celular interna; N. D.- no determinada; N.V.- No viable.Acronyms: MCI.- internal cell mass; N. D.- not determined; N.V.- Not viable.
Diferencias estadísticas entre:Statistical differences between:
1 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a ablación con láser para blastocistos de buena calidad en gelatina: p=0.00001. 1 Direct culture of the blastocyst assisted by the laser technique against laser ablation for good quality blastocysts in gelatin: p = 0.00001.
El método del cultivo directo del blastocisto tiene varias desventajas. Primero, que las células del trofoectodermo proliferan muy rápidamente, reprimiendo el crecimiento de Ia MCI y segundo que existe un riesgo de sobrecrecimiento del trofoectodermo y es habitual que se arrastren células del trofoectodermo cuando se procede a Ia separación de Ia MCI (ver Figuras 3A y 3B). Para salvar esas desventajas, se utilizó Ia técnica de ablación con láser desarrollada recientemente, basada en disparos de láser a las células del trofoectodermo para desprenderse de dichas células no deseadas (ver figuras 3C, 3D y 3E). La eficiencia del aislamiento de MCI sobre MEFs fue prácticamente idéntica entre las técnicas de cultivo directo del blastocisto y Ia de ablación con láser (53% frente a 47%, p≥0,05, ver tabla 1). Sin embargo, aunque esta nueva técnica basada en láser permite una eliminación más limpia del trofoectodermo, solo puede ser aplicada a embriones de buena calidad (figura 3C y tablas 1 y 2), no siendo posible con
embriones de mala calidad (figuras 1D y 1E), en los cuales Ia MCI no es distinguible y no puede, por tanto, ser identificada por el embriólogo.The direct blastocyst culture method has several disadvantages. First, that the trophoctoctoderm cells proliferate very rapidly, repressing the growth of the MCI and second that there is a risk of overgrowth of the trophoctoctoderm and it is common for trophoctoctoder cells to be dragged when the separation of the MCI is carried out (see Figures 3A and 3B). To overcome these disadvantages, the recently developed laser ablation technique was used, based on laser shots to the trophoctoctoder cells to get rid of said unwanted cells (see Figures 3C, 3D and 3E). The efficiency of the isolation of MCI on MEFs was practically identical between the direct culture techniques of the blastocyst and the laser ablation technique (53% versus 47%, p≥0.05, see table 1). However, although this new laser-based technique allows a cleaner removal of the trophoctoctoder, it can only be applied to good quality embryos (Figure 3C and Tables 1 and 2), not being possible with poor quality embryos (figures 1D and 1E), in which the MCI is not distinguishable and cannot, therefore, be identified by the embryologist.
Con el objetivo de de mejorar Ia eficiencia del aislamiento de Ia MCI y de Ia derivación de células troncales embrionarias en comparación con el cultivo directo del blastocisto o con Ia técnica de ablación con láser y también para aprovechar embriones de mala calidad, normalmente descartados, es por Io que se ha desarrollado el procedimiento de Ia invención en dos pasos, basado en Ia fijación del embrión bien a MEFs, bien a gelatina mediante el cultivo directo del blastocisto (figura 3F), seguido de Ia ablación con láser del trofoectodermo (figura 3G y 3H). La figura 3G muestra Ia MCI aislada libre de células de trofoectodermo, mientras que Ia figura 3H muestra los restos del trofoectodermo destruido por los disparos del láser.In order to improve the efficiency of the isolation of the MCI and the derivation of embryonic stem cells compared to the direct culture of the blastocyst or with the laser ablation technique and also to take advantage of poor quality embryos, normally discarded, it is Therefore, the procedure of the invention has been developed in two steps, based on the fixation of the embryo either to MEFs, or to gelatin by direct cultivation of the blastocyst (Figure 3F), followed by the laser ablation of the trophoectoderm (Figure 3G and 3H). Figure 3G shows the isolated MCI free of trophoectoderm cells, while Figure 3H shows the remains of the trophoctoctore destroyed by the laser shots.
Sorprendentemente, cuando se usan embriones de buena calidad, el procedimiento de Ia invención basado en cultivo directo del blastocisto seguido de ablación con láser del trofoectodermo incrementa significativamente Ia eficiencia del aislamiento de Ia MCI sobre MEFs hasta el 100% comparado con el 53% de eficiencia cuando se utiliza el cultivo directo del blastocisto o el 47% cuando se utiliza Ia ablación con láser separadamente. Más aún, sobre gelatina como superficie de crecimiento, este nuevo método desarrollado multiplica por siete Ia eficiencia del aislamiento de Ia MCI (desde el 16% hasta el 100%; p=0,0001) (ver tabla 2).Surprisingly, when good quality embryos are used, the process of the invention based on direct culture of the blastocyst followed by laser ablation of the trophoectoderm significantly increases the efficiency of the isolation of the MCI on MEFs up to 100% compared to 53% efficiency when direct blastocyst culture is used or 47% when laser ablation is used separately. Moreover, on gelatin as a growth surface, this new developed method multiplies by seven the efficiency of the isolation of the MCI (from 16% to 100%; p = 0.0001) (see table 2).
Igual o más importante es que se ha observado que Ia fijación previa de embriones de mala calidad a una mono-capa de MEFs seguida de ablación con láser del trofoectodermo, dio lugar a una tasa de éxito en el aislamiento de Ia MCI significativamente superior (casi el doble, del 100% frente al 66%), cuando se compara con Ia técnica de cultivo directo del blastocisto (ver tabla 1). Sobre gelatina como superficie de crecimiento, el procedimiento de Ia invención originó una ligera mejora de Ia eficiencia del aislamiento de Ia MCI (75% frente al 65%) (ver tabla 2). En conjunto, los datos mostrados indican, que sin importar Ia superficie de crecimiento y Ia calidad de los blastocistos, el procedimiento de Ia invención (cultivo directo
del blastocisto seguido de ablación con láser) es el más eficiente para el aislamiento de MCI.Equally or more importantly, it has been observed that prior fixation of poor quality embryos to a mono-layer of MEFs followed by laser ablation of the trophoectoderm, resulted in a success rate in the isolation of the significantly higher MCI (almost double, 100% versus 66%), when compared with the direct blastocyst culture technique (see table 1). On gelatin as a growth surface, the process of the invention caused a slight improvement in the efficiency of the isolation of the MCI (75% vs. 65%) (see table 2). Together, the data shown indicate that, regardless of the growth surface and the quality of the blastocysts, the process of the invention (direct culture of the blastocyst followed by laser ablation) is the most efficient for the isolation of MCI.
Mejora de Ia eficiencia de la derivación de células troncales embrionarias de ratón mediante cultivo directo del blastocisto ayudado de la técnica láser sin importar Ia calidad del blastocisto. Influencia de Ia superficie de crecimiento.Improvement of the efficiency of the derivation of mouse embryonic stem cells by direct culture of the blastocyst aided by the laser technique regardless of the quality of the blastocyst. Influence of the growth surface.
Entre los embriones de buena calidad, el 33% de las MCIs inicial- mente aisladas, crecieron y dieron lugar a líneas de células troncales embrionarias cuando se uso Ia estrategia de cultivo directo del blastocisto ayudado de Ia técnica láser. Ello contrasta con el 16% (2 veces menos; p=0, 04) de MCIs aisladas que dieron lugar a líneas de células troncales embrionarias obtenido mediante Ia técnica de cultivo directo del blastocisto o con el 6% (6 veces menos; p=0,001) mediante Ia técnica de ablación con láser. Es importante también el aumento de Ia eficiencia observado (50% frente a 33%) con embriones de mala calidad cuando el cultivo del blastocisto directo fue seguido de ablación con láser comparado con el simple cultivo directo del blastocisto (ver tabla 3).Among the good quality embryos, 33% of the initially isolated MCIs grew and gave rise to embryonic stem cell lines when the direct culture strategy of the blastocyst aided by the laser technique was used. This contrasts with 16% (2 times less; p = 0.04) of isolated MCIs that gave rise to embryonic stem cell lines obtained by means of the direct blastocyst culture technique or with 6% (6 times less; p = 0.001) by means of the laser ablation technique. It is also important to increase the observed efficiency (50% versus 33%) with poor quality embryos when the direct blastocyst culture was followed by laser ablation compared to the simple direct blastocyst culture (see table 3).
Cuando se usó gelatina como superficie de crecimiento, se produjo aislamiento con éxito de MCIs (ver tabla 2), pero no se pudo derivar ninguna línea de células troncales embrionarias de ratón, sin importar Ia calidad del blastocisto o el método de aislamiento de Ia MCI utilizado (ver tabla 4).
When gelatin was used as a growth surface, there was successful isolation of MCIs (see table 2), but no line of mouse embryonic stem cells could be derived, regardless of the quality of the blastocyst or the method of isolation of the MCI used (see table 4).
Tabla 3: Eficacia del establecimiento de células troncales embrionarias según Ia calidad de los blastocistos y el método de aislamiento de Ia MCI usando MEFs como superficie de crecimiento.Table 3: Efficacy of the establishment of embryonic stem cells according to the quality of the blastocysts and the method of isolation of the MCI using MEFs as growth surface.
Acrónimos: MCI.- masa celular interna; MEFs.- fibroblastos embrionarios de ratón inactivos; N. V.- No viable Diferencias estadísticas entre: 1 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a cultivo directo del blastocisto para blastocistos de buena calidad: p=0.04.Acronyms: MCI.- internal cell mass; MEFs.- inactive mouse embryonic fibroblasts; N. V.- Not viable Statistical differences between: 1 Direct culture of the blastocyst aided by the laser technique versus direct culture of the blastocyst for blastocysts of good quality: p = 0.04.
2 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a ablación con láser para blastocistos de buena calidad: p=0.00001. 2 Direct culture of the blastocyst aided by the laser technique against laser ablation for blastocysts of good quality: p = 0.00001.
3 Cultivo directo del blastocisto ayudado de Ia técnica láser frente a cultivo directo del blastocisto para blastocistos de mala calidad: p=0.1. 3 Direct culture of the blastocyst aided by the laser technique versus direct culture of the blastocyst for blastocysts of poor quality: p = 0.1.
En conjunto, los datos mostrados indican que Ia combinación del cultivo directo del blastocisto combinado con Ia ablación por láser mejora significativamente Ia eficiencia de Ia derivación de células troncales embrionarias de ratón sin importar Ia calidad del blastocisto, pero dependiendo de Ia superficie de crecimiento utilizada. El procedimiento de Ia invención proporciona una alternativa a Ia inmunocirugía y al cultivo directo del blastocisto, de forma que se pueda llevar a cabo Ia derivación de líneas de células troncales embrionarias aprovechando blastocistos (de ratón y posteriormente de humanos) de mala calidad, frescos y especialmente
congelados, ya que éstos constituyen Ia principal fuente de material de partida para Ia derivación de líneas celulares troncales embrionarias.Together, the data shown indicates that the combination of direct blastocyst culture combined with laser ablation significantly improves the efficiency of mouse embryonic stem cell bypass regardless of the quality of the blastocyst, but depending on the growth surface used. The method of the invention provides an alternative to immunosurgery and the direct culture of the blastocyst, so that the derivation of embryonic stem cell lines can be carried out taking advantage of blastocysts (mouse and later of humans) of poor quality, fresh and especially frozen, since these constitute the main source of starting material for the derivation of embryonic stem cell lines.
Tabla 4: Eficacia del establecimiento de células troncales embrionarias según Ia calidad de los blastocistos y el método de aislamiento de Ia MCI usando gelatina como superficie de crecimiento.Table 4: Efficacy of the establishment of embryonic stem cells according to the quality of the blastocysts and the method of isolation of the MCI using gelatin as a growth surface.
Acrónimos: MCI.- masa celular interna; MEFs.- fibroblastos embrionarios de ratón inactivos; N. V.- No viableAcronyms: MCI.- internal cell mass; MEFs.- inactive mouse embryonic fibroblasts; N. V.- Not viable
Caracterización de líneas de células troncales embrionarias de ratón establecidas mediante Ia técnica de cultivo directo del blastocisto ayudado de Ia técnica láser.Characterization of mouse embryonic stem cell lines established by the direct culture technique of the blastocyst assisted by the laser technique.
Para confirmar que el procedimiento de Ia invención no perjudica las propiedades biológicas intrínsecas de las líneas de células troncales embrionarias derivadas, se han caracterizado las líneas resultantes del cultivo directo del blastocisto ayudado de Ia técnica láser. Estas líneas muestran una morfología típica de células troncales embrionarias (ver figuras 4A y 4B) y expresan marcadores asociados con estado indiferenciado SSEA-1 (Figura 4C) y Oct3/4 (Figura 4D). Para evaluar su potencial de diferenciación in vitro, se formaron cuerpos embrionarios a los cuales se les permitió diferenciarse bajo condiciones que promovieran su diferenciación en tejidos representativos de las tres capas germinales. Como resultado se obtuvo que las líneas de células troncales embrionarias de ratón derivadas mediante Ia técnica de cultivo directo del blastocisto ayudada con láser mostraran una expresión homogénea de los siguientes marcadores:
o alfafetoproteína asociado con endodermo (figura 5B) o actina asociado con mesodermo (figura 5C) y o nestina asociado con ectodermo (figura 5D)To confirm that the process of the invention does not damage the intrinsic biological properties of the derived embryonic stem cell lines, the lines resulting from the direct culture of the assisted blastocyst of the laser technique have been characterized. These lines show a typical embryonic stem cell morphology (see Figures 4A and 4B) and express markers associated with undifferentiated state SSEA-1 (Figure 4C) and Oct3 / 4 (Figure 4D). To assess their potential for differentiation in vitro, embryonic bodies were formed which were allowed to differentiate under conditions that promoted their differentiation into tissues representative of the three germ layers. As a result, it was obtained that the mouse embryonic stem cell lines derived by means of the direct laser-assisted blastocyst culture technique show a homogeneous expression of the following markers: or alpha-fetoprotein associated with endoderm (figure 5B) or actin associated with mesoderm (figure 5C) and or nestin associated with ectoderm (figure 5D)
En conjunto, estos datos demuestran que Ia metodología de cultivo directo del blastocisto ayudada con láser, objeto de Ia presente invención, no compromete ni Ia capacidad de las líneas de células troncales embrionarias de ratón derivadas para mantenerse en estado indiferenciado en cultivo, ni su potencial de diferenciación en las tres capas germinales.Together, these data demonstrate that the direct culture methodology of the laser-assisted blastocyst, object of the present invention, does not compromise the ability of derived mouse embryonic stem cell lines to maintain an undifferentiated state in culture, nor its potential of differentiation in the three germ layers.
Influencia del método utilizado para Ia derivación de líneas de células troncales embrionarias de ratón en el cariotipo de los embriones.Influence of the method used for the derivation of mouse embryonic stem cell lines in the karyotype of the embryos.
El cultivo durante periodos prolongados de células troncales embrionarias se asocia con cambios cariotípicos, quedando por resolver si el procedimiento de derivación de las líneas de células troncales embrionarias es en si mismo responsable de Ia pérdida de estabilidad genética. Se ha analizado si Ia metodología objeto de Ia presente invención usada para Ia derivación de células troncales embrionarias hace a los embriones más vulnerables a inestabilidad genética tras cultivo prolongado. Usando cariotipado convencional, se evaluó Ia frecuencia de aneuploidía de las líneas derivadas usando el método mecánico de cultivo directo con láser o el de ablación con láser.The culture during prolonged periods of embryonic stem cells is associated with karyotypic changes, remaining to be resolved if the procedure of derivation of the embryonic stem cell lines is itself responsible for the loss of genetic stability. It has been analyzed if the methodology object of the present invention used for the derivation of embryonic stem cells makes embryos more vulnerable to genetic instability after prolonged culture. Using conventional karyotyping, the frequency of aneuploidy of the derived lines was evaluated using the mechanical method of direct laser culture or laser ablation.
Las líneas de células troncales embrionarias de ratón establecidas mediante cualquiera de los métodos se mantuvieron diploides tras periodos cortos (12 pases) de cultivo (ver tabla 5 y figuras 6A y 6B). Sin embargo, y sin influencia de Ia técnica de derivación empleada, todas las líneas de células troncales embrionarias adquirieron un cariotipo aneuploide anormal (más de 40 cromosomas) tras periodos prolongados (35 pases) de cultivo (tabla 5 y figuras 6C y 6D). Estos datos sugieren que Ia técnica empleada para Ia derivación de las células troncales embrionarias no hace a los embriones más vulnerables a inestabilidad genómica.
Tabla 5: Frecuencia de aneuploidía en líneas de células troncales embrionarias de ratón derivadas mediante cultivo directo del blastocisto frente a las derivadas mediante ablación con láser.Mouse embryonic stem cell lines established by any of the methods were maintained diploid after short periods (12 passes) of culture (see table 5 and figures 6A and 6B). However, and without influence of the derivation technique used, all embryonic stem cell lines acquired an abnormal aneuploid karyotype (more than 40 chromosomes) after prolonged periods (35 passes) of culture (Table 5 and Figures 6C and 6D). These data suggest that the technique used for the derivation of embryonic stem cells does not make embryos more vulnerable to genomic instability. Table 5: Frequency of aneuploidy in mouse embryonic stem cell lines derived by direct culture of the blastocyst versus those derived by laser ablation.
B) Blastocistós humanosB) Human blastocysts
Los embriones humanos utilizados proceden de parejas sometidas a un ciclo de fecundación "in vitró" (FIV), y que han sido donados a investigación con células madre tras Ia firma de un consentimiento informado. Estos embriones sobrantes se encuentran crioconservados en nitrógeno líquido a -196° C. La tasa de éxito utilizando este tipo de embriones, respecto a evolución a blastocisto y éxito en Ia derivación de células troncales embrionarias humanas (en adelante hESCs), es menor que si se hubieran podido utilizar embriones frescos. Mientras que en experimentos con ratones sí se han podido utilizar embriones frescos, Ia Ley 14/2006 sobre Técnicas de Reproducción Humana Asistida permite sólo Ia utilización de embriones sobrantes congelados. Se ha utilizado para el crecimiento de las colonias una capa de "feeders" humanos, eliminando el uso de gelatina, debido al mal resultado obtenido en experimentos anteriores (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Ligero G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by láser drilling technology enhances the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem. CeIIs. Develop. 2008; 17:255-267).
Obtención de embriones y evolución a blastocistosThe human embryos used come from couples undergoing a cycle of fertilization "in vitró" (IVF), and have been donated to research with stem cells after the signing of an informed consent. These leftover embryos are cryopreserved in liquid nitrogen at -196 ° C. The success rate using this type of embryos, regarding blastocyst evolution and success in the derivation of human embryonic stem cells (hereinafter hESCs), is lower than if Fresh embryos could have been used. While fresh embryos have been able to be used in experiments with mice, Law 14/2006 on Assisted Human Reproduction Techniques allows only the use of frozen embryos left over. A layer of human feeders has been used for the growth of the colonies, eliminating the use of gelatin, due to the poor result obtained in previous experiments (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Light G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by laser drilling technology enhancing the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos : new insights in the derivation of hESC lines. "Stem. CeIIs. Develop. 2008; 17: 255-267). Obtaining embryos and evolution to blastocysts
Los embriones crioconservados donados a investigación por las parejas, o mujer en su caso, sometidos a un ciclo de fecundación "¡n vitro" (FIV), fueron descongelados de acuerdo al método de congelación utilizado al efecto. Todos los embriones que sobrevivieron a Ia descongelación se colocaron en cultivo hasta su evolución al estadio de blastocisto, utilizando los medios G1 y G2 v.5 (Vitrolife, Sweden).Cryopreserved embryos donated to research by couples, or women, where appropriate, underwent a "n vitro" (IVF) fertilization cycle, were thawed according to the freezing method used for this purpose. All embryos that survived thawing were placed in culture until their evolution to the blastocyst stage, using means G1 and G2 v.5 (Vitrolife, Sweden).
58 embriones humanos fueron incluidos en el presente estudio que presta soporte experimental al procedimiento de Ia invención. Fueron descongelados en estadio de división temprana (Día+2, Día+3 postinseminación).58 human embryos were included in the present study that provides experimental support to the method of the invention. They were thawed in the early division stage (Day + 2, Day + 3 post-contamination).
Método de aislamiento de Ia MCI Cultivo directo del blastocisto ayudado de Ia técnica láserMethod of isolation of the MCI Direct culture of the blastocyst assisted by the laser technique
Es el objetivo principal de Ia presente invención. En un intento de mejorar Ia metodología en el proceso de aislamiento de Ia MCI y posterior establecimiento de hESCs a partir de blastocistos expandidos de buena y mala calidad, se ha desarrollado un nuevo método, consistente en dos pasos, i) cultivo directo del blastocisto y, ii) tecnología láser. Con este método, todos los blastocistos humanos fueron tratados con ácido Tyrode para Ia completa disolución de Ia zona pelúcida durante no más de un minuto. Los blastocistos fueron colocados en Ia superficie de crecimiento buscando Ia adhesión de Ia MCI y las células del trofoectodermo. Uno o dos días después, dicha adhesión fue confirmada y se procedió al cambio de medio por medio nuevo y fresco. En torno al día 3 de cultivo, las células del trofoectodermo comenzaron a expandirse, dejando Ia MCI accesible, formando una estructura cupular. En este momento es cuando se utiliza el láser para disparar a todas las células del trofoectodermo que rodean a Ia MCI, dejando esta área adyacente libre de células del trofoectodermo y reduciendo el riesgo de arrastre de dichas células cuando Ia MCI sea subcultivada y transferida a una nueva superficie de crecimiento fresca.
Cultivo de Ia colonia primariaIt is the main objective of the present invention. In an attempt to improve the methodology in the process of isolation of the MCI and subsequent establishment of hESCs from expanded blastocysts of good and poor quality, a new method has been developed, consisting of two steps, i) direct culture of the blastocyst and , ii) laser technology. With this method, all human blastocysts were treated with Tyrode acid for the complete dissolution of the zona pellucida for no more than one minute. The blastocysts were placed on the growth surface seeking the adhesion of the MCI and the trophoctoctoder cells. One or two days later, said adhesion was confirmed and the medium was changed to a new and fresh medium. Around day 3 of culture, the trophoctoctoder cells began to expand, leaving the MCI accessible, forming a cupular structure. At this time it is when the laser is used to shoot all the trophoctoctoder cells surrounding the MCI, leaving this adjacent area free of trophoctoctoder cells and reducing the risk of dragging said cells when the MCI is subcultured and transferred to a New fresh growing surface. Cultivation of the primary colony
Las colonias primarias de las células obtenidas por el procedimiento mencionado fueron cultivadas sobre "feeders" humanos en medio compuesto de Dubelcco's Modified Eagle's Médium (DMEM) Knockout (80%), suero de reemplazo (20%), aminoácidos no esenciales (1%), L- glutamina (1%), 2-mercaptoetanol (0,2%), a 37° C de temperatura y 5% de CO2.The primary colonies of the cells obtained by the aforementioned procedure were grown on human feeders in compound medium of Dubelcco's Modified Eagle's Medium (DMEM) Knockout (80%), replacement serum (20%), non-essential amino acids (1%) , L-glutamine (1%), 2-mercaptoethanol (0.2%), at 37 ° C temperature and 5% CO 2 .
Superficies de crecimientoGrowth surfaces
Para determinar que superficie de crecimiento facilita el aislamiento de Ia MCI y el establecimiento de las hESCs, las colonias primarias fueron cultivadas individualmente sobre "feeders" humanos. La razón de no utilizar para este experimento fibroblastos embrionarios de ratón (MEFs) fue Ia de eliminar cualquier tipo de agente xenobiótico. Además, se eliminó el uso de gelatina como superficie de crecimiento debido al mal resultado que dio en el modelo murino (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Ligero G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by láser drilling technology enhances the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem. CeIIs. Develop. 2008; 17:255-267).To determine which growth surface facilitates the isolation of MCI and the establishment of hESCs, the primary colonies were grown individually on human feeders. The reason for not using mouse embryonic fibroblasts (MEFs) for this experiment was to eliminate any type of xenobiotic agent. In addition, the use of gelatin as a growth surface was eliminated due to the poor result it gave in the murine model (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Lightweight G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by laser drilling technology enhancing the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines . "Stem. CeIIs. Develop. 2008; 17: 255-267).
Resultados obtenidos De los 58 embriones humanos descongelados llegaron al estadio de blastocisto 18 embriones (31 ,0%). Estos blastocistos fueron clasificados de buena o mala calidad atendiendo al grado de expansión del blastocisto, el aspecto del trofoectodermo, así como al aspecto de Ia MCI (Figura 8).Results obtained Of the 58 human embryos thawed 18 embryos arrived at the blastocyst stage (31.0%). These blastocysts were classified as good or bad quality based on the degree of expansion of the blastocyst, the appearance of the trophoctoctoder, as well as the aspect of the MCI (Figure 8).
Sobre Ia base de esta distribución de Ia calidad de los blastocistos, se diseñó Ia estrategia experimental ilustrada en Ia Figura 8 con el propósito de determinar si el procedimiento de cultivo directo del blastocisto ayudado de Ia técnica láser es útil para los siguientes propósitos:
I. mejora de Ia eficiencia del aislamiento de Ia MCI y del establecimiento de células troncales embrionarias yOn the basis of this distribution of the quality of the blastocysts, the experimental strategy illustrated in Figure 8 was designed with the purpose of determining whether the direct culture procedure of the assisted blastocyst of the laser technique is useful for the following purposes: I. improvement of the efficiency of the isolation of the MCI and the establishment of embryonic stem cells and
II. si se podrían utilizar con éxito con blastocistos de baja calidad, los cuales no podrían haberse usado mediante Ia tecnología de ablación con láser debido a que Ia MCI no sería distinguible para el embriólogo y serían normalmente descartados (Figuras 7 y 8).II. if they could be used successfully with low-quality blastocysts, which could not have been used by laser ablation technology because the MCI would not be distinguishable for the embryologist and would normally be discarded (Figures 7 and 8).
Las MCI aisladas se dejaron expandir sobre "feeders" humanos, habiéndose demostrado anteriormente una mejor tasa de éxito sobre esta superficie, comparada con el uso de gelatina (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Ligero G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by láser drilling technology enhances the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem. CeIIs. Develop. 2008; 17:255-267).The isolated MCIs were allowed to expand on human feeders, having previously demonstrated a better success rate on this surface, compared with the use of gelatin (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A , Barroso A, Cabrera C, Light G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by laser drilling technology enhancing the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines. "Stem. CeIIs. Develop. 2008; 17: 255-267).
Mejora de Ia eficiencia del aislamiento de Ia MCI mediante cultivo directo del blastocisto ayudado de Ia técnica láserImprovement of the efficiency of the isolation of the MCI by direct culture of the blastocyst aided by the laser technique
De los 18 blastocistos obtenidos tras Ia descongelación de 58 embriones, 5 fueron clasificados como de buena calidad (27,8%), y 13 blastocistos fueron clasificados como de mala calidad (72,2%). Todos los blastocistos fueron liberados de Ia zona pelúcida mediante el uso de acido Tyrode, y colocados individualmente sobre una capa de "feeders" humanos para el crecimiento de Ia colonia primaria. Tras los días pertinentes de cultivo se procedió a aislar Ia MCI mediante el método de aislamiento objeto de Ia invención. De los 5 embriones de buena calidad, se pudo aislar Ia MCI en 4 ocasiones (80%), mientras que con blastocistos de mala calidad, en 8 de 13 de blastocistos se pudo realizar este procedimiento (62%). Si se comparan estos resultados con los obtenidos en el experimento realizado con blastocistos de ratón, se observa que Ia tasa de éxito utilizando blastocistos humanos ha bajado respecto a los blastocistos murinos (80% vs. 100% en blastocistos de buena calidad, y 62% vs. 100% en blastocistos de mala
calidad) cuando se utilizó como superficie de crecimiento "feeders" humanos. Si por el contrario, se comparan los resultados obtenidos con blastocistos humanos con los obtenidos con blastocistos murinos, pero utilizando con estos últimos gelatina como superficie de crecimiento, se observa que con los de buena calidad Ia tasa también baja (80% vs. 100%). Sin embargo, en blastocistos de mala calidad Ia calidad solamente baja mínimamente (62% vs. 75%) (Tabla 1).Of the 18 blastocysts obtained after thawing 58 embryos, 5 were classified as of good quality (27.8%), and 13 blastocysts were classified as of poor quality (72.2%). All blastocysts were released from the zona pellucida through the use of Tyrode acid, and placed individually on a layer of human feeders for the growth of the primary colony. After the relevant days of cultivation, the MCI was isolated by means of the isolation method object of the invention. Of the 5 embryos of good quality, the MCI could be isolated 4 times (80%), while with blastocysts of poor quality, 8 of 13 of blastocysts could perform this procedure (62%). If these results are compared with those obtained in the experiment performed with mouse blastocysts, it is observed that the success rate using human blastocysts has decreased with respect to murine blastocysts (80% vs. 100% in good quality blastocysts, and 62% vs. 100% in bad blastocysts quality) when human feeders were used as growth surface. If, on the contrary, the results obtained with human blastocysts are compared with those obtained with murine blastocysts, but using these last gelatin as a growth surface, it is observed that with those of good quality the rate also falls (80% vs. 100% ). However, in blastocysts of poor quality the quality only decreases minimally (62% vs. 75%) (Table 1).
Tabla 1 : Eficiencia del aislamiento de Ia MCI basado en Ia calidad del blastocisto y en el método de aislamiento de Ia MCI usando blastocistos murinos y humanos.Table 1: Efficiency of the isolation of the MCI based on the quality of the blastocyst and the method of isolation of the MCI using murine and human blastocysts.
Acrónimos: MCI.- masa celular interna; MEFs.- fibroblastos embrionarios de ratón inactivos.Acronyms: MCI.- internal cell mass; MEFs.- inactive mouse embryonic fibroblasts.
Independientemente de Ia dificultad que supone poder conseguir un gran número de embriones para investigación, muy por debajo de Ia cantidad que se puede conseguir en ratones, no cabe duda de que Ia calidad de los embriones congelados es peor que si se utilizan frescos, en Ia mayoría de los casos (figuras 7 A y 7B). Es por esto, que en este experimento se han tenido que destinar los embriones humanos autorizados para este proyecto hacia el método de aislamiento de Ia MCI más eficaz, según Io demostrado en modelo murino, así como utilizar para su crecimiento una superficie de "feeders" humanos. Esta elección se ha debido a que el método del cultivo directo del blastocisto tiene varias desventajas. Primero, que las células del trofoectodermo proliferan muy rápidamente,
reprimiendo el crecimiento de Ia MCI y segundo que existe un riesgo de sobrecrecimiento del trofoectodermo y es habitual que se arrastren células del trofoectodermo cuando se procede a Ia separación de Ia MCI. Para salvar esas desventajas, se ha utilizado en ratones (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Ligero G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by láser drilling technology enhances the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem. CeIIs. Develop. 2008; 17:255-267), y en humanos (Turetsky T, Aizenman E, Gil Y, Weinberg N, Shufaro Y, Revel A, Laufer N, Simón A, Abeliovich D, Reubinoff BE. "Laser- assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis." Hum. Reprod. 2008 ¡23:46-53), Ia técnica de ablación con láser, basada en disparos de láser a las células del trofoectodermo para desprenderse de dichas células no deseadas. Sin embargo, aunque esta nueva técnica basada en láser permite una eliminación más limpia del trofoectodermo, solo puede ser aplicada a embriones de buena calidad (figura 7C), no siendo posible con blastocistos de mala calidad (figura 7D), en los cuales Ia MCI no es distinguible y no puede, por tanto, ser identificada por el embriólogo.Regardless of the difficulty of being able to achieve a large number of embryos for research, well below the amount that can be achieved in mice, there is no doubt that the quality of frozen embryos is worse than if fresh ones are used, in Ia most cases (figures 7 A and 7B). This is why, in this experiment, the human embryos authorized for this project have been destined for the most effective method of isolation of the MCI, as demonstrated in the murine model, as well as using a feeder surface for its growth humans. This choice has been due to the fact that the direct blastocyst culture method has several disadvantages. First, that trophoctoctoderm cells proliferate very rapidly, repressing the growth of the MCI and second that there is a risk of overgrowth of the trophoctoctoderm and it is usual for the trophoctoctoder cells to be dragged when the separation of the MCI is carried out. To overcome these disadvantages, it has been used in mice (Cortes JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martinez-Ramirez A, Barroso A, Cabrera C, Light G, Montes R, Rubio R, Nieto A, Menendez P. "Whole-blastocyst culture followed by laser drilling technology enhancing the efficiency of ICM isolation and ESC derivation from good and poor-quality mouse embryos: new insights in the derivation of hESC lines." Stem. CeIIs. Develop. 2008; 17: 255-267), and in humans (Turetsky T, Aizenman E, Gil Y, Weinberg N, Shufaro Y, Revel A, Laufer N, Simon A, Abeliovich D, Reubinoff BE. "Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. "Hum. Reprod. 2008 ¡23: 46-53), the laser ablation technique, based on laser shots to the trophoctoctoder cells to get rid of said unwanted cells. However, although this new laser-based technique allows a cleaner removal of the trophoctoctoderm, it can only be applied to embryos of good quality (Figure 7C), not being possible with blastocysts of poor quality (Figure 7D), in which the MCI It is not distinguishable and cannot, therefore, be identified by the embryologist.
Con el objetivo de mejorar Ia eficiencia del aislamiento de Ia MCI y de Ia derivación de células troncales embrionarias en comparación con el cultivo directo del blastocisto o con Ia técnica de ablación con láser y también para aprovechar embriones de mala calidad, normalmente descartados, es por Io que se ha desarrollado el procedimiento de Ia invención en dos pasos, basado en Ia obtención de un blastocisto a partir de un embrión descongelado en estadio de división temprana (figura 9A, 9B y 9C), Ia fijación a una superficie de "feeders" humanos (figura 9D), seguido de Ia ablación con láser del trofoectodermo (figura 9E). La figura 9F muestra Ia MCI aislada libre de células de trofoectodermo.
Consideraciones relativas al origen de los embriones en relación con el Art. 5.1c de Ia Lev de PatentesIn order to improve the efficiency of the isolation of the MCI and the derivation of embryonic stem cells compared to the direct culture of the blastocyst or with the laser ablation technique and also to take advantage of poor quality embryos, normally discarded, it is by Io that the procedure of the invention has been developed in two steps, based on obtaining a blastocyst from a thawed embryo in early division stage (Figure 9A, 9B and 9C), the fixation to a surface of "feeders" human (Figure 9D), followed by laser ablation of the trophoctoctoderm (Figure 9E). Figure 9F shows the isolated MCI free of trophoectoderm cells. Considerations regarding the origin of embryos in relation to Art. 5.1c of the Patent Law
Mientras que las células madre de modelos animales y las células madre somáticas y germinales humanas no generan ningún tipo de problema ético-moral, son las hESCs las que llevan inherentes este tipo de controversias, ya que el aislamiento de Ia masa celular interna de los blastocistos supone Ia destrucción de estos. Por ello, es necesario un gran control legislativo, tanto a nivel nacional como europeo, que regule su obtención y aplicación en salud. La legislación en España respecto al uso de embriones humanos sobrantes de ciclos de fecundación "in vitro" (FIV) está actualmente regulada por Ia Ley 14/2006, sobre técnicas de reproducción asistida, por el Real Decreto 1301/2006, sobre células y tejidos humanos, y por Ia Ley 14/2007, sobre investigación biomédica. La pareja sometida a un ciclo FIV puede dar un destino final a aquellos embriones sobrantes que se encuentren crioconservados en nitrógeno líquido, independientemente del tiempo de congelación, siempre bajo consentimiento informado de Ia pareja. Estos embriones llevan en muchas ocasiones más de 5 años congelados sin que Ia pareja, o mujer en su caso, los hayan reclamado. Los diferentes destinos posibles que pueden darse a los embriones sobrantes crioconservados son: a) Su utilización por Ia propia mujer o cónyuge. b) La donación con fines reproductivos. c) La donación con fines de investigación. d) El cese de su conservación sin otra utilización. Respecto a Ia utilización de embriones con fines de investigación, sólo se autorizará si se atiene a varios requisitos: a) Posesión del consentimiento informado. b) Que el preembrión no se haya desarrollado in vitro más allá de 14 días después de Ia fecundación. c) Que Ia investigación se realice en centros autorizados. d) Que Ia investigación se realice en base a un proyecto debidamente presentado y autorizado por las autoridades competentes.
e) Que se especifiquen las posibles relaciones de interés entre centros.While the stem cells of animal models and human somatic and germ stem cells do not generate any ethical-moral problem, it is the hESCs that carry this type of controversy, since the isolation of the internal cell mass of the blastocysts supposes the destruction of these. Therefore, great legislative control is necessary, both at national and European level, which regulates its obtaining and application in health. The legislation in Spain regarding the use of human embryos left over from "in vitro" (IVF) fertilization cycles is currently regulated by Law 14/2006, on assisted reproduction techniques, by Royal Decree 1301/2006, on cells and tissues humans, and by Law 14/2007, on biomedical research. The couple undergoing an IVF cycle can give a final destination to those remaining embryos that are cryopreserved in liquid nitrogen, regardless of freezing time, always with the informed consent of the couple. These embryos have been frozen for more than 5 years without the couple, or woman in their case, having claimed them. The different possible destinations that can be given to cryopreserved embryos are: a) Its use by the woman or spouse. b) Donation for reproductive purposes. c) Donation for research purposes. d) The cessation of its conservation without other use. Regarding the use of embryos for research purposes, it will only be authorized if it meets several requirements: a) Possession of informed consent. b) That the pre-embryo has not developed in vitro beyond 14 days after fertilization. c) That the investigation be carried out in authorized centers. d) That the investigation be carried out based on a project duly submitted and authorized by the competent authorities. e) That the possible relations of interest between centers be specified.
Esta legislación deja a España en un término medio de permisividad, englobándola en el grupo de países donde está permitido investigar con embriones sobrantes de ciclos de FIV para derivar hESCs (Canadá,This legislation leaves Spain in a medium term of permissiveness, encompassing it in the group of countries where it is allowed to investigate with embryos left over from IVF cycles to derive hESCs (Canada,
Holanda, Australia, Suecia), lejos del grupo de países donde está prohibido utilizar embriones para investigación con hESCs (Irlanda, Austria, Noruega), aunque un peldaño por debajo del grupo de países donde se pueden generar embriones con fines únicos de investigación (Reino Unido, Bélgica, Israel, Singapur).Holland, Australia, Sweden), far from the group of countries where it is prohibited to use embryos for research with hESCs (Ireland, Austria, Norway), although a step below the group of countries where embryos can be generated for single research purposes (Kingdom United, Belgium, Israel, Singapore).
Alternativamente, se podrían derivar blastocistos a los cuales someter al procedimiento objeto de Ia invención a partir de técnicas que no comprometen Ia viabilidad del embrión, tales como:Alternatively, blastocysts could be derived to which to submit to the process object of the invention from techniques that do not compromise the viability of the embryo, such as:
- zigotos triples no viables (ver patente europea EP 1516925) - transferencia nuclear alterada [Meissner y Jánisch; Nature- non-viable triple zygotes (see European patent EP 1516925) - altered nuclear transfer [Meissner and Jánisch; Nature
439, 212-215 ; (2006)]439, 212-215; (2006)]
- single cell embryo biopsy [Chung et al.; Nature 439, 216- 219; (2006)]
- single cell embryo biopsy [Chung et al .; Nature 439, 216-219; (2006)]
Claims
1.- Procedimiento para el aislamiento de Ia masa celular interna en blastocistos de mamíferos que incluye una etapa de ablación mediante láser del trofoectodermo de dichos blastocistos y caracterizado porque previamente a dicha etapa de ablación mediante láser los blastocistos se someten a cultivo directo en placa durante un periodo de tiempo de al menos 72 horas.1.- Procedure for the isolation of the internal cell mass in mammalian blastocysts that includes a laser ablation stage of the trophoctoctoder of said blastocysts and characterized in that prior to said laser ablation stage the blastocysts are subjected to direct plate culture during a period of time of at least 72 hours.
2.- Procedimiento para el aislamiento de Ia masa celular interna en blastocistos de mamíferos según Ia reivindicación 1 , caracterizado porque los blastocistos son de mamíferos no humanos.2. Procedure for the isolation of the internal cell mass in mammalian blastocysts according to claim 1, characterized in that the blastocysts are of non-human mammals.
3.- Procedimiento para el aislamiento de Ia masa celular interna en blastocistos de mamíferos, según Ia reivindicación 2, caracterizado porque los blastocistos son de ratón.3. Procedure for the isolation of the internal cell mass in mammalian blastocysts, according to claim 2, characterized in that the blastocysts are mouse.
4.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-3, caracterizado porque el cultivo directo previo a Ia ablación mediante láser incluye las siguientes sub-etapas: a) cultivo de los blastocistos recuperados de los oviductos en medio estándar, particularmente medio de cultivo G2 V.lll (Vitrolife, Sweden) a 370C y con 6% de CO2 durante un periodo de tiempo comprendido entre 24 y 48 horas. b) tratamiento de los blastocistos cultivados en Ia sub-etapa anterior para conseguir una completa eliminación de Ia zona pelúcida, c) colocación de los blastocistos tratados en Ia etapa anterior sobre una superficie de cultivo y posterior adhesión del trofoectodermo y Ia masa celular interna de los blastocistos a dicha superficie. d) mantenimiento de los blastocistos sobre dicha superficie y en un medio estándar de cultivo durante un periodo de tiempo comprendido entre 24 y 48 horas. e) refresco del medio de cultivo y mantenimiento de los blastocistos en el mismo durante un nuevo periodo de tiempo comprendido entre 24 y 48 horas.4. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 1-3, characterized in that the direct culture prior to laser ablation includes the following sub-stages: a) culture of the blastocysts recovered from the oviducts in standard medium, particularly G2 V.lll culture medium (Vitrolife, Sweden) at 37 0 C and with 6% CO 2 for a period of time between 24 and 48 hours. b) treatment of the blastocysts cultured in the previous sub-stage to achieve a complete elimination of the zona pellucida, c) placement of the blastocysts treated in the previous stage on a culture surface and subsequent adhesion of the trophoctoctoder and the internal cell mass of the blastocysts to said surface. d) maintenance of the blastocysts on said surface and in a standard culture medium for a period of time between 24 and 48 hours. e) refreshment of the culture medium and maintenance of the blastocysts in it for a new period of time between 24 and 48 hours.
5.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-4, caracterizado porque Ia eliminación de Ia zona pelúcida se lleva acabo mediante tratamiento con ácido Tyrode o enzima pronasa durante un periodo de tiempo comprendido entre 30 y 60 segundos.5. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 1-4, characterized in that the removal of the pellucid zone is carried out by treatment with Tyrode acid or pronase enzyme for a period of time between 30 and 60 seconds.
6.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-4, caracterizado porque Ia eliminación de Ia zona pelúcida se lleva a cabo por eclosión espontánea del blastocisto.6. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 1-4, characterized in that the elimination of the pellucid zone is carried out by spontaneous hatching of the blastocyst.
7.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-6, caracterizado porque Ia superficie de cultivo sobre Ia cual se colocan los blastocistos tras Ia eliminación de Ia zona pelúcida es una placa recubierta con MEFs (Mouse embryonic fibroblasts).7. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 1-6, characterized in that the culture surface on which the blastocysts are placed after the removal of the pellucid zone is a plate covered with MEFs (Mouse embryonic fibroblasts).
8.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-6, caracterizado porque Ia superficie de cultivo sobre Ia cual se colocan los blastocistos tras Ia eliminación de Ia zona pelúcida es una placa recubierta de gelatina.8. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 1-6, characterized in that the culture surface on which the blastocysts are placed after the removal of the pellucid zone is a gelatin coated plate.
9.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-8, caracterizado porque el medio en el cual se mantienen los blastocistos sobre Ia superficie de cultivo es medio DMEM (Dubelcco's Modified Eagle's Médium) suplementado con suero fetal bovino al 20%, L-glutamina al 1%, aminoácidos no esenciales al 0,1 mM, 2000 lU/ml de factor inhibidor de leucemia de ratón, β- mercaptoetanol 0,1 mM, 50 U/ml de penicilina y 50 μg/ml de estreptomicina.9. Method for the isolation of inner cell mass in mammalian blastocysts according to claims 1-8, wherein the medium in which on the surface blastocysts culture is maintained DMEM (Dulbecco 's Modified Eagle Medium) supplemented with 20% bovine fetal serum, 1% L-glutamine, non-essential amino acids at 0.1 mM, 2000 lU / ml of mouse leukemia inhibitor factor, 0.1 mM β-mercaptoethanol, 50 U / ml of penicillin and 50 μg / ml of streptomycin.
10.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 1-3, caracterizado porque Ia etapa de ablación mediante láser del trofoectodermo de los blastocistos, a realizar inmediatamente tras Ia etapa de cultivo previo, se lleva a cabo con un dispositivo de diodo láser de emisión en infrarrojo a una frecuencia óptica (longitud de onda) de 1 ,48 μm.10. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 1-3, characterized in that the laser ablation stage of the blastocyst trophoectoderm, to be performed immediately after the previous culture stage, is carried out with an infrared emission laser diode device at an optical frequency (wavelength) of 1.48 μm.
11.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según Ia reivindicación 10, caracterizado porque para Ia ablación mediante láser del trofoectodermo de los blastocistos se realizan entre 20 y 100 disparos.11. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claim 10, characterized in that between 20 and 100 shots are carried out by laser ablation of the blastocyst trophoectoderm.
12.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según Ia reivindicación 11 , caracterizado porque tras Ia ablación con láser del trofoectodermo de los blastocistos, se produce el establecimiento de las líneas celulares embrionarias de ratón a partir de Ia masa celular interna aislada de los blastocistos sometidos al procedimiento.12. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claim 11, characterized in that after laser ablation of the blastocyst trophoctoctoder, the establishment of mouse embryonic cell lines from the cell mass occurs internally isolated from the blastocysts undergoing the procedure.
13.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según Ia reivindicación 1 , caracterizado porque los blastocistos de mamíferos son blastocistos humanos.13. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claim 1, characterized in that the mammalian blastocysts are human blastocysts.
14.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según Ia reivindicación 13, caracterizado porque el cultivo directo previo a Ia ablación mediante láser incluye las siguientes sub-etapas: a) descongelación de embriones humanos en estadio de división temprana y cultivo hasta Ia evolución a blastocisto utilizando medios G1 y G2 v.5 (Vitrolife, Sweden). b) tratamiento de los blastocistos cultivados en Ia subetapa anterior para conseguir una completa eliminación de Ia zona pelúcida. c) colocación de los blastocistos tratados en Ia etapa anterior sobre una superficie de cultivo y posterior adhesión del trofoectodermo y Ia masa celular interna de los blastocistos a dicha superficie. d) mantenimiento de los blastocistos sobre dicha superficie y en un medio estándar de cultivo durante un periodo de tiempo comprendido entre 24 y 48 horas. e) refresco del medio de cultivo y mantenimiento de los blastocistos en el mismo durante un nuevo periodo de tiempo comprendido entre 24 y 48 horas.14. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claim 13, characterized in that the direct culture prior to laser ablation includes the following sub-stages: a) thawing of human embryos in the early division stage and culture until evolution to blastocyst using means G1 and G2 v.5 (Vitrolife, Sweden). b) treatment of the blastocysts cultured in the anterior sub-stage to achieve a complete elimination of the zona pellucida. c) placing the blastocysts treated in the previous stage on a culture surface and subsequent adhesion of the trophoctoctoderm and the internal cell mass of the blastocysts to said surface. d) maintenance of the blastocysts on said surface and in a standard culture medium for a period of time between 24 and 48 hours. e) refreshment of the culture medium and maintenance of the blastocysts in it for a new period of time between 24 and 48 hours.
15.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según Ia reivindicaciones 13 y 14, caracterizado porque Ia eliminación de Ia zona pelúcida se lleva acabo mediante tratamiento con ácido Tyrode o enzima pronasa durante un periodo de tiempo comprendido entre 30 y 60 segundos.15.- Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 13 and 14, characterized in that the removal of the pellucid zone is carried out by treatment with Tyrode acid or pronase enzyme for a period of time between 30 and 60 seconds.
16.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 13 y 14, caracterizado porque Ia eliminación de Ia zona pelúcida se lleva a cabo por eclosión espontánea del blastocisto.16. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 13 and 14, characterized in that the elimination of the pellucid zone is carried out by spontaneous hatching of the blastocyst.
17.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 13 a 16, caracterizado porque Ia superficie de cultivo sobre Ia cual se colocan los blastocistos tras Ia eliminación de Ia zona pelúcida es una placa recubierta con "feeders" humanos.17. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 13 to 16, characterized in that the culture surface on which the blastocysts are placed after the removal of the pellucid zone is a plate coated with feeders. humans.
18.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicaciones 13-17, caracterizado porque el medio en el cual se mantienen los blastocistos sobre Ia superficie de cultivo es medio DMEM (Dubelcco's Modified Eagle's Médium) Knockout (80%), suero de reemplazo (20%), aminoácidos no esenciales (1%), L- glutamina (1%), 2-mercaptoetanol (0,2%), a 37° C y 5% de CO2.18. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claims 13-17, characterized in that the means in which the blastocysts are maintained on the surface culture is DMEM (Dulbecco 's Modified Eagle Medium) Knockout (80%), serum replacement (20%), non - essential (1%) amino acids, L - glutamine (1%), 2-mercaptoethanol (0.2 %), at 37 ° C and 5% CO 2 .
19.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según las reivindicación 13, caracterizado porque Ia etapa de ablación mediante láser del trofoectodermo de los blastocistos, a realizar inmediatamente tras Ia etapa de cultivo previo, se lleva a cabo con un dispositivo de diodo láser de emisión en infrarrojo a una frecuencia óptica (longitud de onda) de 1 ,48 μm.19. Procedure for the isolation of internal cell mass in mammalian blastocysts according to claim 13, characterized in that the laser ablation stage of the blastocyst trophoectoderm, to be performed immediately after the previous culture stage, is carried out with a infrared emission laser diode device at an optical frequency (wavelength) of 1.48 μm.
20.- Procedimiento para el aislamiento de masa celular interna en blastocistos de mamíferos según Ia reivindicación 19, caracterizado porque para Ia ablación mediante láser del trofoectodermo de los blastocistos se realizan entre 20 y 100 disparos. 20.- Procedure for the isolation of internal cell mass in mammalian blastocysts according to claim 19, characterized in that between 20 and 100 shots are carried out by laser ablation of the blastocyst trophoectoderm.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ESP200701625 | 2007-06-01 | ||
| ES200701625A ES2326577B1 (en) | 2007-06-01 | 2007-06-01 | PROCEDURE FOR THE INSULATION OF INTERNAL CELL MASS IN BLASTOCIST OF NON-HUMAN MAMMALS. |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008145788A1 true WO2008145788A1 (en) | 2008-12-04 |
Family
ID=40074604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/ES2008/000377 WO2008145788A1 (en) | 2007-06-01 | 2008-05-23 | Method for isolation of the inner cell mass in mammal blastocysts |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2326577B1 (en) |
| WO (1) | WO2008145788A1 (en) |
-
2007
- 2007-06-01 ES ES200701625A patent/ES2326577B1/en not_active Withdrawn - After Issue
-
2008
- 2008-05-23 WO PCT/ES2008/000377 patent/WO2008145788A1/en active Application Filing
Non-Patent Citations (6)
Also Published As
| Publication number | Publication date |
|---|---|
| ES2326577B1 (en) | 2010-07-08 |
| ES2326577A1 (en) | 2009-10-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2588088C (en) | Derivation of embryonic stem cells | |
| Cao et al. | Isolation and culture of primary bovine embryonic stem cell colonies by a novel method | |
| Tanaka et al. | Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model | |
| Zhang et al. | Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation | |
| Lim et al. | In Vitro Culture‐Induced Pluripotency of Human Spermatogonial Stem Cells | |
| Lee et al. | Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis | |
| CN105062958A (en) | Composition for embryonic stem cell culture and application thereof | |
| JP2007516720A (en) | Embryonic stem cell line and method for producing the same | |
| CN103114075A (en) | Cattle trophoderm stem cell system establishment method | |
| WO2008145788A1 (en) | Method for isolation of the inner cell mass in mammal blastocysts | |
| Klimanskaya | Embryonic stem cells from blastomeres maintaining embryo viability | |
| De Los Angeles et al. | Highly efficient derivation of pluripotent stem cells from mouse preimplantation and postimplantation embryos in serum-free conditions | |
| Intawicha et al. | Derivation and characterization of putative embryonic stem cells from cloned rabbit embryos | |
| González et al. | Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods | |
| Desai et al. | Vitrification of mouse embryo-derived ICM cells: a tool for preserving embryonic stem cell potential? | |
| Cheng et al. | Blastocoel volume is related to successful establishment of human embryonic stem cell lines | |
| Cao et al. | Isolation and culture of bovine embryonic stem cells | |
| CN109609446B (en) | A kind of culture medium and method for separating and culturing rabbit embryonic stem cells | |
| Veiga et al. | Selection of embryos for stem cell derivation: can we optimize the process | |
| CN119592502A (en) | Male embryonic stem cells from a BALB/c mouse strain and its application | |
| Pall et al. | Establishment of an embryonic stem cell line from blastocyst stage mouse embryos | |
| Takeuchi et al. | Harvesting of embryonic stem cells aimed at embryo conservation | |
| Moon et al. | Efficiency of equilibrium and vitrification solution of mesenchymal stem cells obtained from human amnion | |
| Cho et al. | Derivation of mouse ES Cells from isolated blastomeres in culture media supplemented with LIF | |
| Zhang et al. | Effective Derivation and Manipulation of Mouse Embryonic Stem Cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08775405 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 08775405 Country of ref document: EP Kind code of ref document: A1 |