Hathaitip Sritanaudomchai

Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Mitochondrial gene replacement in primate offspring and embryonic stem cells

Mitochondria are found in all eukaryotic cells and contain their own genome (mitochondrial DNA or mtDNA). Unlike the nuclear genome, which is derived from both the egg and sperm at fertilization, the mtDNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin. Mutations in mtDNA contribute to a diverse range of currently incurable human diseases and disorders. To establish preclinical models for new therapeutic approaches, we demonstrate here that the mitochondrial genome can be efficiently replaced in mature non-human primate oocytes (Macaca mulatta) by spindle–chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg. The reconstructed oocytes with the mitochondrial replacement were capable of supporting normal fertilization, embryo development and produced healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infants born so far originated from the spindle donors whereas mtDNA came from the cytoplast donors. No contribution of spindle donor mtDNA was detected in offspring. Spindle replacement is shown here as an efficient protocol replacing the full complement of mitochondria in newly generated embryonic stem cell lines. This approach may offer a reproductive option to prevent mtDNA disease transmission in affected families.

Epigenetic Reprogramming by Somatic Cell Nuclear Transfer in Primates

We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X‐inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte‐specific factors. STEM CELLS 2009;27:1255–1264

Methylation Status of Imprinting Centers for H19/IGF2 and SNURF/SNRPN in Primate Embryonic Stem Cells

Embryonic stem cells (ESCs) hold promise for cell and tissue replacement approaches to treating human diseases based on their capacity to differentiate into a wide variety of somatic cells and tissues. However, long‐term in vitro culture and manipulations of ESCs may adversely affect their epigenetic integrity, including imprinting. We have recently reported aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ESC lines, whereas SNRPN and NDN were normally imprinted and expressed predominantly from the paternal allele. The dysregulation of IGF2 and H19 that is associated with tumorigenesis in humans may result from improper maintenance of allele‐specific methylation patterns at an imprinting center (IC) upstream of H19. To test this possibility, we performed methylation analysis of several monkey ESC lines by genomic bisulfite sequencing. We investigated methylation profiles of CpG islands within the IGF2/H19 IC harboring the CTCF‐6 binding site. In addition, the methylation status of the IC within the promoter/exon 1 of SNURF/SNRPN known as the Prader‐Willi syndrome IC was examined. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analyzed ESC lines, whereas the SNURF/SNRPN IC was differentially methylated, consistent with monoallelic expression.

CDX2 in the formation of the trophectoderm lineage in primate embryos

The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.

Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells

BACKGROUND Parthenogenetic embryonic stem cells (PESCs) may have future utilities in cell replacement therapies since they are closely related to the female from which the activated oocyte was obtained. Furthermore, the avoidance of parthenogenetic development in mammals provides the most compelling rationale for the evolution of genomic imprinting, and the biological process of parthenogenesis raises complex issues regarding differential gene expression. METHODS AND RESULTS We describe here homozygous rhesus monkey PESCs derived from a spontaneously duplicated, haploid oocyte genome. Since the effect of homozygosity on PESCs pluripotency and differentiation potential is unknown, we assessed the similarities and differences in pluripotency markers and developmental potential by in vitro and in vivo differentiation of homozygous and heterozygous PESCs. To understand the differences in gene expression regulation between parthenogenetic and biparental embryonic stem cells (ESCs), we conducted microarray analysis of genome-wide mRNA profiles of primate PESCs and ESCs derived from fertilized embryos using the Affymetrix Rhesus Macaque Genome array. Several known paternally imprinted genes were in the highly down-regulated group in PESCs compared with ESCs. Furthermore, allele-specific expression analysis of other genes whose expression is also down-regulated in PESCs, led to the identification of one novel imprinted gene, inositol polyphosphate-5-phosphatase F (INPP5F), which was exclusively expressed from a paternal allele. CONCLUSION Our findings suggest that PESCs could be used as a model for studying genomic imprinting, and in the discovery of novel imprinted genes.

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.

Erratum: Human embryonic stem cells derived by somatic cell nuclear transfer (Cell (2013) 153 (1228-1238))

Masahito Tachibana, Paula Amato, Michelle Sparman, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Hong Ma, Eunju Kang, Alimujiang Fulati, Hyo-Sang Lee, Hathaitip Sritanaudomchai, Keith Masterson, Janine Larson, Deborah Eaton, Karen Sadler-Fredd, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard L. Stouffer, Don Wolf, and Shoukhrat Mitalipov* *Correspondence: mitalipo@ohsu.edu http://dx.doi.org/10.1016/j.cell.2013.06.042

BmKn-2 scorpion venom peptide for killing oral cancer cells by apoptosis.

Scorpion venom peptides recently have attracted attention as alternative chemotherapeutic agents that may overcome the limitations of current drugs, providing specific cytotoxicity for cancer cells with an ability to bypass multidrug-resistance mechanisms, additive effects in combination therapy and safety. In the present study, BmKn-2 scorpion venom peptide and its derivatives were chosen for assessment of anticancer activities. BmKn-2 was identified as the most effective against human oral squamous cells carcinoma cell line (HSC-4) by screening assays with an IC50 value of 29 μg/ml. The BmKn-2 peptide killed HSC-4 cells through induction of apoptosis, as confirmed by phase contrast microscopy and RT-PCR techniques. Typical morphological features of apoptosis including cell shrinkage and rounding characteristics were observed in treated HSC-4 cells. The results were further confirmed by increased expression of pro-apoptotic genes such as caspase-3, -7, and -9 but decrease mRNA level of anti-apoptotic BCL-2 in BmKn-2 treated cells, as determined by RT-PCR assay. In summary, the BmKn-2 scorpion venom peptide demonstrates specific membrane binding, growth inhibition and apoptogenic activity against human oral cancer cells.

The scorpion venom peptide BmKn2 induces apoptosis in cancerous but not in normal human oral cells

AIM This study aimed to investigate the mechanism of the induction of apoptosis of human oral cancer cells by the scorpion venom peptide BmKn2. METHODS Human oral squamous carcinoma cells (HSC4), mouth epidermoid carcinoma cells (KB), human normal gingival cells (HGC) and dental pulp cells (DPC) were treated with BmKn-2 peptide for 24h. Cell viability was determined by the MTT assay. Apoptosis was assessed using phase contrast microscopy, by propidium iodide (PI) staining to assess nuclear morphology and by Annexin V staining. Apoptotic signaling pathways were investigated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting. RESULTS BmKn-2 showed potent cytotoxic effects towards both HSC4 and KB cells with the associated induction of apoptosis. The cells showed distinct morphological changes, nuclear disintegration and an increase in the number of Annexin V-positive cells. Interestingly, at concentrations which kill cancerous cells, BmKn-2 did not affect cell viability or mediate the induction of apoptosis in normal HGC or DPC. Induction of apoptosis by BmKn-2 in HSC4 and KB cells was associated with the activation of tumor suppress p53. Pro-apoptotic BAX expression was increased, whereas antiapoptotic BCL-2 expression was decreased in BmKn-2 exposed HSC4 and KB cells. BmKn-2 treated-oral cancer cells showed distinct upregulation of initiator caspase-9, with no effect on caspase-8 expression. Increased expression levels of executor caspases-3 and -7 were also found in treated cells for both oral cancers. CONCLUSION This study has suggested for the first time that BmKn-2 exerts selective cytotoxic effects on human oral cancer cells by inducting apoptosis via a p53-dependent intrinsic apoptotic pathway. BmKn-2 peptide originally derived from a natural source shows great promise as a candidate treatment for oral cancer, with minimal effects on healthy tissue.