Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells

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Abstract

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells1,2,3, holding promise for autologous cell replacement therapy4,5. Although methods to induce pluripotent stem cells from somatic cells by transcription factors6 are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported7,8,9,10,11, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

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Figure 1: Developmental potential of somatic cell nuclear transfer oocytes.
Figure 2: Chromosome condensation and spindle assembly after somatic cell nuclear transfer.
Figure 3: Derivation of diploid NT-ES cells from adult somatic cells.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data are available at GEO under accession numbers GSE54849 and GSE54876.

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Acknowledgements

This research was supported the New York Stem Cell Foundation (NYSCF) and a New York State Stem Cell Science (NYSTEM) IIRP Award no. C026184, and the Russell Berrie Foundation Program in Cellular Therapies of Diabetes. We thank S. Mitalipov for helpful discussions and providing reagents, S. Micucci for counting cells in S-phase, and Z. Hall for critical reading of the manuscript. D.E. is a NYSCF-Robertson Investigator.

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Authors

Contributions

M.V.S. supervised the research oocyte donation program and retrieved oocytes. D.E. designed, performed and interpreted nuclear transfer experiments, derived ES cells with M.Y., and wrote the paper with input from all authors. M.Y. performed statistical analysis, M.Y. and B.J. performed stem cell characterization and differentiation, L.C.B. performed neuronal differentiation, I.S. and N.B. performed gene expression analysis, M.W.N. assisted with calcium experiments, D.H.K. performed data analysis, D.P. assisted in nuclear transfer experiments, R.W.P. collected developmental data of IVF embryos, M.F. made the skin biopsy, E.G. coordinated human subjects research, R.S.G. wrote the IRB protocol, R.L.L. contributed project planning, S.L.S. created the environment specifically for this work.

Corresponding author

Correspondence to Dieter Egli.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Efficiency of parthenogenetic development beyond the cleavage stage.

Shown is the percentage of oocytes giving rise to stem cell lines, blastocysts but no stem cell lines, and morulae as the percentage of the number of oocytes cleaved. Data are from manuscript references12,14 and displayed here in a direct comparison. The number of oocytes used is indicated above the column. The number of repeats, with oocytes from different donors, is indicated in parenthesis. Statistical analysis using Chi-square test was performed by comparing the total number of cells formed in each condition. Morulae were assigned 15 cells, blastocysts or blastocysts that gave rise to stem-cell lines 30 cells, reflecting the estimated minimal cell count for each group.

Extended Data Figure 2 Development to the blastocyst stage and transcriptional activation of the transferred genome.

a, Blastocyst derived after nuclear transfer of a BJ fibroblast genome (neonatal foreskin fibroblasts). b, Blastocysts derived after nuclear transfer of an adult skin fibroblast genome. c, Three different nuclear transfer ES cell outgrowths. Time post blastocyst plating is indicated. Scale bar, 10 µm.

Extended Data Figure 3 Fluorescence imaging with the calcium-responsive dye Fluo-4.

a, Human oocytes were incubated in medium containing Fluo-4 for 30 min, imaged for fluorescence, and concentrated Sendai virus was added below the plasma membrane. Shown are two oocytes for each condition or time point. Note that in the absence of calcium in the medium, fluorescence did not increase, whereas a small increase in fluorescence seems to occur in calcium-containing medium. Time point after addition of the virus is indicated. b, Incubation of a human oocyte in 3 µM of the calcium ionophore ionomycin as a positive control. Scale bar, 10 µm.

Extended Data Figure 4 Somatic cell nuclear transfer in the absence of calcium.

a, Immunochemistry to determine chromosome condensation and histone phosphorylation after transfer of a somatic cell at interphase. Scale bar, 5 µm. b, High-quality blastocysts obtained after nuclear transfer with the manipulations conducted in the absence of calcium. Scale bar, 10 µm.

Extended Data Figure 5 Effect of FBS on blastocyst morphology and ES cell derivation.

a, Blastocyst generated by somatic cell nuclear transfer in the presence or absence of FBS. b, Inner cell mass 6 days post plating. Arrows point to laser marks used to ablate the remaining trophectoderm cells. Scale bar, 20 µm.

Extended Data Figure 6 Characterization of NT-ES cells from male foreskin BJ fibroblasts.

ac, Karyotypes and pluripotency marker expression in three NT-ES cell lines derived from male foreskin BJ fibroblasts. The somatic donor cell used for transfer carries a GFP transgene. Scale bar, 50 µm.

Extended Data Figure 7 Differentiation of NT-ES cell lines made from male foreskin BJ fibroblasts.

Embryoid bodies and teratomas are shown. Scale bar, 50 µm.

Extended Data Figure 8 Retrospective analysis of the developmental potential of nuclear transfer oocytes.

Shown is the percentage of oocytes developing beyond the cleavage stage, as percentage of eggs progressing beyond the 1-cell stage. Because oocytes of a donor were used to compare two different conditions, if for a particular comparison the added number of oocyte donors exceeds 18, it indicates that these conditions were tested in parallel using oocytes of the same donor. The total number of oocytes used for analysis remained constant. a, Average of the 154 oocytes of 18 donors. The total number of 154 oocytes is not equal to the number of oocytes donated by the 18 donors, but is the number of oocytes used for the study of developmental potential after somatic cell nuclear transfer. b, Analysis with regard to factors relevant to the hormonal treatment of oocyte donor. c, Factors relevant to the manipulation. *Blastocysts generated without caffeine treatment also contained no FBS in the culture medium. Because of the effect of FBS on inner cell mass morphology (Extended Data Fig. 5), FBS is likely the more relevant factor. d, Analysis regarding cell source and use of FBS for culture. n.s., non significant. Statistical analysis using Chi-square test was performed by comparing the total number of cells formed in each condition. Morulae were assigned 15 cells, blastocysts or blastocysts that gave rise to stem-cell lines 30 cells, reflecting the estimated minimal cell count for each group.

Extended Data Table 1 Complete list of samples used in global gene expression analyses
Extended Data Table 2 Short tandem repeat (STR) profiles of nuclear transfer ES cell lines

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Yamada, M., Johannesson, B., Sagi, I. et al. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells. Nature 510, 533–536 (2014). https://doi.org/10.1038/nature13287

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