Abstract
BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.
Highlights
To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke
Our results indicate that a self-renewable and homogenous population of human neural stem cells (hNSCs), SD56, was derived from Human embryonic stem cells (hESCs) using defined media supplemented with a specific combination of growth factors
The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog
Summary
To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. ESCs were cultured in a defined media supplemented with morphogens or growth factors [5,6,7] or cultured under conditions that promote ‘‘rosettes’’, structures morphologically similar to the developing neural tube [8,9] This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10,11,12]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]) These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo
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