Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies.

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.

Nuclear transfer for full karyotyping and preimplantation diagnosis for translocations.

Preimplantation genetic diagnosis (PGD) for chromosomal disorders is currently performed by interphase fluorescence in-situ hybridization (FISH) analysis. This technique is known to have limitations for detecting some translocations and complete karyotyping, so visualization of chromosomes in single cells is required to improve the PGD accuracy for chromosomal disorders. To achieve this, single blastomeres were fused with enucleated or intact mouse zygotes, followed by fixing the resulting heterokaryons at the metaphase of the first cleavage division, or treating them with okadaic acid to induce premature chromosome condensation. This method allowed a significant improvement in the accuracy of testing both maternally- and paternally-derived translocations. In all, 437 blastomeres were tested, achieving full karyotyping in as many as 383 (88%), making it possible to pre-select only normal embryos or those with balanced chromosomal complements for transfer. Overall, PGD for translocations was applied in 94 clinical cycles, resulting in 66 transfers and 20 (30.3%) clinical pregnancies, with healthy deliveries of 15 children. Fifty-two of these cycles were performed using a nuclear transfer (conversion) technique, which resulted in 38 transfers of balanced or normal embryos, demonstrating that the technique is accurate and reliable for karyotyping single blastomeres for PGD of translocations.