br Resource table Resource details Following their mechanica

Resource table: Resource details Following their mechanical isolation from day 7 blastocysts, the inner cell masses (ICM) were seeded individually onto the feeder layers. After 2days of incubation, most of the ICMs were found attached to the feeder cells. The cultures were allowed to grow for 8–10 days, with medium changes every alternate day, to develop into primary embryonic stem cell (ESC) colonies. The primary colonies were subcultured onto fresh feeders to produce ESC lines. Out of the initial 20–30 primary colonies, only four to five made it up to 10 passages and beyond. Our main purpose for establishment of these XX-ESC lines was to develop strategies and study mechanisms for their differentiation into oocytes. All the putative cell lines obtained were characterized for various stem cell markers by RT-PCR and immunocytochemistry, according to the already established criteria for human ESC characterization (Heins et al., 2004). All of the putative buESC colonies had a characteristic dome-shaped morphology of buESC colonies and were strongly positive for alkaline phosphatase. (Fig. 1). RT-PCR was positive for various stem cell markers like OCT4, NANOG, SOX2, c-MYC, REX-1, STAT3, FOXD3 and TELOMERASE (Fig. 2). Immunocytochemical analysis for stem cell markers carried out for intact colonies as well as monolayer adherent isosafrole showed positive expression of various pluripotency and self renewal-associated transcription factors like OCT4, NANOG, SOX2 and FOXD3 (Fig. 3) as well as of ESC surface markers like SSEA1, SSEA4, TRA-1-60, TRA-1-81 and CD-90 (Fig. 4). The cell lines karyotyped at various passages (passage 10, 30, and 40) revealed normal chromosomal content throughout the culture interval. In all of the 20 spreads evaluated, karyotyping revealed normal and exact metaphase euploid content of a female murrah buffalo (Bubalus bubalis) (48 +XX) genome (Fig. 5). Upon spontaneous differentiation in hanging drop (HD) cultures, the colonies from all three stem cell lines differentiated into EBs. RT-PCR analysis of the EBs showed expression of ectodermal (CYTOKERATIN8 and NF68), mesodermal (BMP4 and MSX 1), and endodermal (AFP and GATA4) markers (Fig. 6). None of these markers was expressed in undifferentiated colonies. Immunocytochemistry also revealed expression of CYTOKERATIN8, BMP4, and GATA4 in the EBs when examined at day 8 of the culture period, whereas no such protein expression was detected in control (undifferentiated) colonies (Fig.7).
Materials and methods
Resource table: Resource details Human embryonic stem cell (hESC) line chHES-419 was derived from abnormal blastocyst donated by Marfan syndrome patient who underwent preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed a heterozygous deletion mutation, c.3536delA, of FBN1 in the cells. The deletion caused a frameshift at amino acid 1179 (glutamine) resulting in early termination after 25 abnormal amino acid residues (Q1179Rfs*25), indicating the pathogenic effect of this mutation (Fig. 1A). The result is consistent with the proband. The cells displayed pluripotent cell morphology (Fig. 1B), expressed pluripotency related genes (Fig. 1C) and were positive for a set of pluripotent markers OCT3/4, NANOG, TRA-1-60 and TRA-1-81 as well as alkaline phosphatase (Fig. 1D). The cells had the capability to differentiate into the derivatives from all the three germ layers both in vitro (Fig. 1E) and in vivo (Fig. 1F). In addition, this line maintained the normal karyotype 46, XX during long-term culture (Fig. 1G).
Materials and methods
Acknowledgments This work was supported by grants from the National Basic Research Program of China (973 program 2011CB964901 and 2012CB944901), the National Natural Science Foundation of China (81222007 and 31101053) and Central South University innovation project of research (2014zzts290). We thank the genetic laboratory and IVF team of the Reproductive & Genetic Hospital of CITIC-Xiangya for their assistance.