Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

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These results supported initiation of a Phase I clinical trial in patients with sensorimotor complete thoracic SCI.

Oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem (hES) cells have been reported to remyelinate axons and improve locomotor function in a rodent model of spinal cord injury. Although remyelination would be expected to have a beneficial effect in spinal cord injury, neurotrophic factor expression may also contribute to functional recovery. Neurotrophic factors could impact the survival of axotomized neurons, as well as promote axonal regeneration in interrupted conduction pathways. This study demonstrates that hES cell-derived OPCs express functional levels of midkine, hepatocyte growth factor (HGF), activin A, transforming growth factor-beta2 (TGF-beta2), and brain-derived neurotrophic factor (BDNF), proteins with reported trophic effects on neurons. The neurotrophic activity of hES cell-derived OPCs is further demonstrated by stimulatory effects on neurite outgrowth of adult rat sensory neurons in vitro.