Figure 1. Comparison of Normal Development with Development during Reproductive Cloning and Therapeutic Cloning. During normal development (Panel A), a haploid (1n) sperm cell fertilizes a haploid oocyte to form a diploid (2n) zygote that undergoes cleavage to become a blastocyst embryo. Blastocysts are implanted in the uterus and ultimately give rise to an animal. During reproductive cloning (Panel B), the diploid nucleus of an adult donor cell is introduced into an enucleated oocyte, which after artificial activation divides into a cloned blastocyst. On transfer into surrogate mothers, a few of the cloned blastocysts will give rise to a clone. In contrast, therapeutic cloning (Panel C) requires the explantation of cloned blastocysts in culture to yield a line of embryonic stem cells that can potentially differentiate in vitro into any type of cell for therapeutic purposes.

During presentation, please touch the figure and wait. You will find that the cells can beat. NTU1 human ES cell form cardiomyocyte-like cells after in vitro differentiation in adherent culture

Cumulative percentage of EBs (embryoid body) containing spontaneously contracting areas as a function of the number of days after plating of the EB. Functional analysis of contracting area within the EBs. (a) Calcium transients of human ES cell–derived cardiomyocytes as determined by fura-2 fluorescence. A typical calcium transient is shown on the left, and continuous recording is shown on the right. Tp, time to peak; Tt, total transient time; T1/2, time to half-peak relaxation. (b) Typical extracellular electrophysiological recordings from different areas of the EB. Note the presence of a sharp and slow component. R, ratio.

a, Analysis of amphetamine-stimulated rotations in animals grafted with neurons derived from wild-type (WT) (n = 10) or Nurr1 ES cells (n = 15) and sham controls (n = 18). b−e, Non-pharmacological evaluation of the animals grafted with either WT or Nurr1 ES cells (mean   s.e.m.). b, Nine weeks after grafting and one week after the last injection of amphetamine, spontaneous turning behaviour was evaluated for 5 min. c, The results in the adjusting step test are expressed as a percentage of the lesioned side relative to the number of steps with the non-lesioned paw. d, In the paw-reaching test, the number of pellets eaten with the lesioned paw were normalized by the total number of pellets eaten during the 7-day test period. e, In the cylinder test, the use of each limb was measured when rearing and landing. The percentage of use of the lesioned-side limb relative to the total number of landings after rearing is expressed, Asterisk, P < 0.05; double asterisk, P < 0.001, compared with sham group.

Advanced follicle like form in the next two weeks but the majority degenerate upon further cultivation.

Fig. 5. Transplantation of ES-derived germ cells into adult testis. (a) At 5–6 weeks after transplantation, host testes and transplants were stained with 5-Bromo-4-chloro-3-indolyl  -D-galactoside (X-Gal). (b) A magnified view of the transplant. (c) A representative view of seminiferous tubules in a section of the transplant. (d) A section of the transplant involving unpurified ES cells and embryonic gonadal cells. (e) A transplant of gonadal cells on their own. (Scale bars = 100 µm.) Fig. 6. (A) Sections of wild-type (a–c) testicular tubules and transplant (d–j) tubules double-stained with anti- -gal and affinity-purified anti-HSC70t antibodies. Shown is nuclear staining (a, d, and g), anti- -gal staining (b, e, and h), and anti-HSC70t staining (c, f, and i). (j) Higher magnification (x6) of a stained transplant tubule and merged image of g–i. Arrowheads in g and h indicate positions of Sertoli and myoid cell. (Bar in a for a–f is 50 µm and in j for g–j is 20µm.) (B) Detection of the knock-in allele in genomic DNA extracted from Mvh-lacZ knock-in ES cells (lane 1), sperm from a lacZ-positive transplant (lane 2), and a wild-type seminiferous tubule (lane 3). Primer pairs detecting lacZ (a), Neo (c), and Sry (d) genes were used for PCR (30 cycles; 94°C, 58°C, and 72°C at 1 min each). (b) Southern blot of the PCR product hybridized with labeled lacZ probe. (e) Photograph of sperm purified from transplant seminiferous tubules.

Reference 2 (original paper Rideout Cell 2002;109:17-27, described in the next slide) Figure 2. Nuclear transfer embryonic stem (NT ES) cells can be created by the transfer of a somatic cell nucleus into an enucleated oocyte. ES cells cultured from the ICM of a blastocyst created by NT can either be manipulated for transplantation or can undergo gene therapy before transplantation for the treatment of genetic disorders.

Figure 1. Scheme for Therapeutic Cloning Combined with Gene and Cell Therapy. A piece of tail from a mouse homozygous for the recombination-activating gene 2 (Rag2) mutation was removed and cultured. After fibroblast-like cells grew out, they were used as donors for nuclear transfer by direct injection into enucleated MII oocytes using a Piezoelectric-driven micromanipulator. Embryonic stem (ES) cells isolated from the NT-derived blastocysts were genetically repaired by homologous recombination. After repair, the ntES cells were differentiated in vitro into embryoid bodies (EBs), infected with the HoxB4iGFP retrovirus, expanded, and injected into the tail vein of irradiated, Rag2-deficient mice.