Reprogramming within hours following nuclear transfer into mouse but not human zygotes - PubMed (original) (raw)

Alice E Chen, Genevieve Saphier, Justin Ichida, Claire Fitzgerald, Kathryn J Go, Nicole Acevedo, Jay Patel, Manfred Baetscher, William G Kearns, Robin Goland, Rudolph L Leibel, Douglas A Melton, Kevin Eggan

Affiliations

Reprogramming within hours following nuclear transfer into mouse but not human zygotes

Dieter Egli et al. Nat Commun. 2011.

Abstract

Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation.

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Figures

Figure 1

Figure 1. Donation of human zygotes for stem cell research

a, embryos donated for stem cell research. PN= pronucleus, ND= not determined b, Mitotic entry time of human zygotes (n=107) in hours after thaw. Nuclear envelope breakdown of at least one of the two pronuclei was defined as the time point of mitotic entry. Some zygotes were mitotic at the time of thaw. c, mitotic spindle formation. Human zygote 30 minutes after nuclear envelope breakdown including brightfield image, microtubule immunohistochemistry (arrowheads point to the centrosome at both poles of the spindle) and spindle birefringence. d-f, asynchrony in nuclear envelope breakdown. d, Hours between the breakdown of the first pronuclear envelope and the second. e, Zygote with asynchronous pronuclear envelope breakdown. Numbers indicate the time from the breakdown of the first nuclear envelope. The location of mitotic chromatin is circled. f, 1PN zygote stained for phosphorylation of Ser 27 on Histone H3, a marker of mitosis.

Figure 2

Figure 2. Somatic cell nuclear transfer into human zygotes

a, Schematic of nuclear transfer into human zygotes. b, zygote at 2PN stage in the presence of nocodazole. Size bar applies to all panels except where otherwise indicated. c, zygote at mitosis in the presence of nocodazole. Maternal and paternal haploid genomes are circled. d, paternal and maternal haploid genomes removed from the zygote. e, Somatic cell nuclear transfer (green nucleus marked by H2B:GFP) by fusion, f, chromosome condensation, g, and after exit from mitosis. Time post exit from mitosis is indicated. **h,**spindle assembly i, chromosomes aligned at metaphase. j, cleavage, k, 6-cell stage, l, morula-like, on d4 post fertilization. Size bar: 25μm.

Figure 3

Figure 3. Developmental potential and ZGA after nuclear transfer into human zygotes

a, Developmental potential of control IVF human zygotes (black columns, n=28), zygotes after nuclear transfer (blue columns, n=53), and zygotes cultured in the presence of alpha-amanitin (red columns, n=23) displayed as the percentage of cells developing to and beyond the indicated developmental stage. b, GFP expression after human nuclear transfer. c, Transgene reactivation after mouse nuclear transfer. Shown are nuclear transfer cells 2 days after transfer into mouse zygotes. d, Genome activation after human SCNT. Venn diagrams of genes upregulated >5-fold in the indicated groups, e, maternal mRNA degradation after human SCNT. Venn diagram of genes with transcript levels reduced by a factor of 5 or more in the indicated groups. NT=nuclear transfer, ZGA=zygotic genome activation. Size bar: 25μm.

Figure 4

Figure 4. Transcriptional reprogramming within hours after mouse nuclear transfer

a, Oct4::GFP reprogramming after somatic cell nuclear transfer into mouse zygotes. b, Number of Oct4-GFP+ colonies during mouse iPS generation. Kinetics of Oct4::GFP reactivation in somatic cells after transduction with retroviruses carrying the reprogramming factors Oct4, Sox2, Klf4 and c-Myc. Day 8 is the 8th day after the first exposure of somatic cells to viral vectors. c,d, ZGA and reprogramming 22h after nuclear transfer into mouse zygotes. c, Venn diagram of transcripts elevated at the 2-cell stage. d, cluster diagram of the global gene expression profile after nuclear transfer into oocytes or zygotes. * from ref. . Size bar 10μm.

Figure 5

Figure 5. Chromosome condensation is required for development and reprogramming after nuclear transfer into mouse zygotes

a, schematic for nuclear transfer into prometaphase and b, corresponding images. Nuclei of fibroblasts at interphase are transferred into a zygote at prometaphase of mitosis. Chromosomes are marked with the red fluorescent fusion protein H2B-cherry. Shown is the mitotic progression after nuclear transfer. Time indicates the hours after nuclear transfer. Note the condensation of chromosomes and their separation into two groups, followed by formation of 2 pronuclei after inhibition of cytokinesis and entry into interphase. c, removal of the genome from a zygote at anaphase. d, Nuclear morphology after transfer of interphase nuclei. Small inset: a somatic donor cell before transfer. Note that somatic donor chromatin is not condensed. e, nuclear remodeling is slow and requires about 1 day to restructure the nucleus into a large blastomere-like morphology. f, cluster analysis of gene expression after mouse SCNT. g, Venn diagram of transcripts elevated 22-24h after nuclear transfer at anaphase. h, development to morula and blastocyst stage (as % of transferred). Size bar 10μm.

Figure 6

Figure 6. Failure to initiate transcription is not caused by karyotypic abnormalities

a, Induction of aneuploidy in human zygotes. Schematic showing development after suppression of cleavage at the first mitosis. b, Development after suppression of cleavage at the first mitosis. Arrows point to a multipolar spindle as detected by microtubule birefringence. Immunohistochemistry at anaphase of mitosis shows the asymmetric segregation of chromatin to three poles (outer surface of the cleaving cell is outlined). Final panel: cleavage directly to 4 cells. NT=nuclear transfer. Days indicated the time post IVF. c, Clustering of the transcriptome of the indicated samples. Size bar 25μm.

Figure 7

Figure 7. Nuclear transfer into human polyspermic zygotes

a, Immunocytochemistry of dispermic human zygotes at mitosis. Arrowheads point to pericentrin positive centrosomes, arrows to tubulin positive sperm tails. b, Entry of polyspermic zygotes into mitosis. c, Nuclear transfer into human polyspermic zygotes. Genome removed at mitosis, fusion with a somatic donor cell and chromosome condensation of the somatic cell genome 2h post transfer are shown. d, Cleavage and development after nuclear transfer into polyspermic zygotes. Time indicates the days post fertilization. The arrow points to a birefringent spindle.

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