Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies |
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| Authors: | Sergey Malchenko Jianping Xie Maria de Fatima Bonaldo Elio F. Vanin Bula J. Bhattacharyya Abdelhak Belmadani Guifa Xi Vasily Galat William Goossens Richard E.B. Seftor Tadanori Tomita John Crispino Richard J. Miller Martha C. Bohn Mary J.C. Hendrix Marcelo B. Soares |
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| Affiliation: | 1. Cancer Biology and Epigenomics Program, Ann & Robert H Lurie Children''s Hospital of Chicago Research Center, Department of Pediatrics, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA;2. Developmental Biology Program, Ann & Robert H Lurie Children''s Hospital of Chicago Research Center, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA;3. Division of Hematology/Oncology, Department of Pediatrics, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA;4. Neurobiology Program, Ann & Robert H. Lurie Children''s Hospital of Chicago Research Center, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA;5. Microscopy and Imaging Facility, Ann & Robert H. Lurie Children''s Hospital of Chicago Research Center, Chicago, IL, USA;6. Molecular Pharmacology & Biological Chemistry, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA;g Division of Pediatric Neurosurgery, Ann & Robert H Lurie Children''s Hospital of Chicago Research Center, Ann & Robert H Lurie Children''s Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA;h Falk Brain Tumor Center, Ann & Robert H Lurie Children''s Hospital of Chicago Research Center, Ann & Robert H Lurie Children''s Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA;i Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA |
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| Abstract: | In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. |
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| Keywords: | hESCs, human embryonic stem cells hiPSC, human induced pluripotent stem cell NOD-SCID, non-obese diabetes-severe combined immunodeficiency EB, embryoid bodies NEC, primitive neuroectoderm NP, neural progenitors RNSC, Rosette neural stem cell line GFP, green fluorescent protein ACSF, artificial celebrospinal fluid RG, radial glia BLBP, brain-lipid-binding protein GFAP, glial fibrillary acidic protein TH, tyrosine hydroxylase |
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