Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001.

The number of human embryonic stem cell (hESC) lines available to federally funded U.S. researchers is currently limited. Thus, determining their basic characteristics and disseminating these lines is important. In this report, we recovered and expanded the earliest available cryopreserved stocks of the BG01, BG02, and BG03 hESC lines. These cultures exhibited multiple definitive characteristics of undifferentiated cells, including long-term self-renewal, expression of markers of pluripotency, maintenance of a normal karyotype, and differentiation to mesoderm, endoderm, and ectoderm. Each cell line exhibited a unique genotype and human leukocyte antigen (HLA) isotype, confirming that they were isolated independently. BG01, BG02, and BG03 maintained in feederfree conditions demonstrated self-renewal, maintenance of normal karyotype, and gene expression indicative of undifferentiated pluripotent stem cells. A survey of gene expression in BG02 cells using massively parallel signature sequencing generated a digital read-out of transcript abundance and showed that this line was similar to other hESC lines. BG01, BG02, and BG03 hESCs are therefore independent, undifferentiated, and pluripotent lines that can be maintained without accumulation of karyotypic abnormalities.

Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.