Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord

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Abstract

Lineage-restricted progenitors of the central nervous system (CNS) are not readily expandable because their mitotic competence is limited. Here we used retroviral overexpression of human telomerase reverse transcriptase (hTERT) to immortalize progenitors from human fetal spinal cord. The hTERT-immortalized cells divided in basic fibroblast growth factor (bFGF) expressed high telomerase activity, and gave rise to phenotypically restricted subpopulations of either glia or neurons. The latter included a prototypic line, hSC11V-TERT, that gave rise only to neurons. These included both chx10+ interneurons and Islet1+/Hb9+/ChAT+ motor neurons; the latter were recognized by green fluorescent protein (GFP) driven by the Hb9 enhancer. The neurons were postmitotic and achieved electrophysiologic competence. Upon xenograft to both fetal rat brain and injured adult spinal cord, they matured as neurons and survived for 6 months, with no evident tumorigenesis. The cells have survived >168 doublings in vitro, with karyotypic normalcy and without replicative senescence. hTERT overexpression thus permits the generation of progenitor lines able to give rise to phenotypically restricted neurons.

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Figure 1: hTERT transduction yielded phenotypically restricted cell lines.
Figure 2: Immortalized lines had high levels of telomerase and long telomeres.
Figure 3: hSC11V-TERT cells generate spinal interneurons and motor neurons.
Figure 4: hSC11V-TERT cells gave rise to mature neurons.
Figure 5: hSC11V-TERT cells retained mitotic and karyotypic stability.
Figure 6: Transplanted hSC11V-TERT-generated neurons are able to achieve widespread integration and phenotypical-functional maturation in injured spinal cord.

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Acknowledgements

Supported by Project ALS and the Christopher Reeve Paralysis Foundation. H.M.K is supported by the Abby Rockefeller Mauze Medical Scientist Fellowship, The Iris L. and Leverett S. Woodworth Medical Scientist Fellowship and NIGMS GM07739. We thank Tom Jessell for the Hb9 9 kb promoter, H. Okano and A. Kawaguchi for the hsp68 promoter, Greg Caporaso for assistance in the TRAP assay, Fraser Sim for his advice and expertise in qPCR, Takahiro Takano for assistance in calcium imaging, and Li Jiang for assistance with patch clamp analysis. We are also grateful to Melissa Kujawski for technical assistance, Brad Poulos of the Fetal Tissue Resource Bank of the Albert Einstein College of Medicine and Geron Corp. for providing the retroviral pBABE-hTERT vector.

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Correspondence to Steven A Goldman.

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M.C. is an employee of Geron.

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Roy, N., Nakano, T., Keyoung, H. et al. Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord. Nat Biotechnol 22, 297–305 (2004) doi:10.1038/nbt944

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