Human somatic cell nuclear transfer using adult cells.

Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.

Reprogramming of human somatic cells using human and animal oocytes.

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc>4; p<0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.

Human somatic cell nuclear transfer.

Human Somatic Cell Nuclear Transfer (hSCNT) is a required procedure before derivation of autologous embryonic stem cells (ESC) for clinical treatment. The debate between ethical concerns and the potential for clinical application is still ongoing. Currently, the milestone of deriving hSCNT blastocysts has been achieved. The developmental mechanism for SCNT is similar to the developmental mechanism for fertilized embryos without sperm fertilization. The hSCNT procedure consists of enucleating an oocyte and replacing with a donor somatic cell nucleus; this procedure is more difficult than a parthenogenetic procedure. The difficulties of SCNT involve the cell cycle synchronization between the donor nucleus and arrested ooplasm, suboptimal activation of reconstructed "zygotes", suboptimal culture condition, and incomplete genetic remodeling/epigenetic modification. There are protocols intended to overcome the difficulties, but no center has derived hSCNT ESC yet.