Identification and characterization of neuronal precursors and their progeny from human fetal tissue

Abstract

We have examined primary human neuronal precursors (HNPs) from 18-22-week-old fetuses. We showed that E-NCAM/MAP2/β-III tubulin-immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament-associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage-gated ion channels, and ligand-gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Tα1 tubulin promoter was E-NCAM/β-III tubulin-immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population. Keywords stem cells; differentiation; electrophysiologyMultipotent human neural stem cells (NSCs) have been isolated from fetal human forebrain and spinal cord (Buc-Caron, 1995;Sabate et al., 1995;Svendsen et al., 1996Svendsen et al., , 1998 ChalmersRedman et al., 1997;Johansson et al., 1999; Vescovi et al., 1999a,b;Carpenter et al., 1999;Quinn et al., 1999;Piper et al., 2000) as well as the adult human subependymal zone and hippocampus (Kukekov et al., 1999; Roy et al., 2000a,b). Furthermore, phenotypically restricted lineages have also been reported for human brain tissue. These include glial restricted precursors (GRPs), which have been isolated from adult human brain tissue (Scolding et al., 1999;Roy et al., 1999), and recent reports have suggested the existence of neuronal restricted precursors (NRPs) in both fetal (Raymon et al., 1999;Li et al., 2000) and adult (Roy et al., 2000a,b;Wang et al., 2000) human tissue.NSCs or neuroepithelial precursor cells (NEPs) can generate neurons in vitro (Carpenter et al., 1999;Piper et al., 2000) as well as in vivo (Svendsen et al., 1998;Vescovi et al., 1999b). In vitro, neurons derived from less committed, NEP-like cell populations develop slowly and do ★ Correspondence to: Mary T. Lucero, PhD, Department of Physiology, 410 Chipeta Way, Room 155, Salt Lake City, UT 84108-1297., mary.lucero@m.cc.utah.edu. NIH Public AccessAuthor Manuscript J Neurosci Res. Author manuscript; available in PMC 2010 November 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript not mature fully until after 70 days under differentiating conditions (Chalmers-Redman et al., 1997). Once differentiated, NEP-derived neuronal cells acquired Na + and K + channels but did not develop the ability to fire action potentials despite prolonged culture periods (>3 weeks; Piper et al., 2000). These data suggested that, in human cells, neuronal maturation may require factors other than those described for r...

Human Cortical Neurons Originate from Radial Glia and Neuron-Restricted Progenitors

“…In LeX Ϫ cultures, neurospheres were less likely to form both in mice (Capela and Temple, 2006) and humans (this study), consistent with the notion that they contained more lineagerestricted progenitors. Finally, the heterogeneity of human cortical progenitor cell population, suggested here, is well correlated with results from other laboratories on human fetal stem cells (Carpenter et al, 2001;Suslov et al, 2002;Messina et al, 2003;Kim et al, 2006) or human fetal forebrain (Piper et al, 2001).…”

“…Comparable proliferating cells labeled with neuronal markers were identified in both the human fetal (Piper et al, 2001, Howard et al, 2006 and murine cortex (Haubensak et al, 2004;Gal et al, 2006).…”

Functional Neural Development from Human Embryonic Stem Cells: Accelerated Synaptic Activity via Astrocyte Coculture

“…For this, primary fetal cultures (Kato et al, 1985;Erkman et al, 1989;Kerkovich et al, 1999), expanded neural progenitors derived from fetal brain tissues (Chalmers-Redman et al, 1997; Svendsen et al, 1997Svendsen et al, , 1998Carpenter et al, 1999;Piper et al, 2001), and immortalized neuronal cell lines (Li et al, 2000) have been the primary models for monitoring human neuronal development. These methods have provided viable neurons capable of physiological maturation, but the temporal development of these neurons varies with tissue age, in vitro expansion, and culturing method (Piper et al, 2001). In contrast, human embryonic stem (hES) cells, capable of differentiating into all cell types (Thomson et al, 1998;Reubinoff et al, 2000), allow the systematic functional evaluation of neural development under highly reproducible conditions.…”

Molecular and Morphological Heterogeneity of Neural Precursors in the Mouse Neocortical Proliferative Zones

“…However, little is known about the cellular and molecular determinants generating this diversity. Numerous studies have suggested that the ventricular zone (VZ) stem cells generate committed neuronal, glial, and bipotential progenitors, each restricted to the production of one or more types of postmitotic cells (Maric et al, 2000;McCarthy et al, 2001;Piper et al, 2001;Cai et al, 2002b;Liu et al, 2002;Shen et al, 2002;Maric et al, 2003;. Indeed, multipotent radial glial cells (RGCs) and distinct committed neuronal and glial progenitors have been identified in the human and monkey VZ (Levitt et al, 1981(Levitt et al, , 1983Ostenfeld and Svendsen, 2004;Zecevic, 2004).…”