Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum

doi:10.1016/0306-4522(94)90452-9

Neuroscience

Volume 58, Issue 4, February 1994, Pages 753-763

https://doi.org/10.1016/0306-4522(94)90452-9 Get rights and content

Abstract

P19 embryonal carcinoma cells are pluripotent and can be efficiently induced to differentiate in culture into neurons and astroglia by brief treatment with retinoic acid. Retinoic acid-treated P19 cells survive after grafting into the adult rat striatum and differentiate into neurons and glia within the transplantation site. No tumours develop from the grafted cells which continue to express foreign genes that had been transfected into the parental P19 cells. The neurons in these grafts express a variety of neurotransmitters similar to those formed in retinoic acid-treated P19 cell cultures and they mature to acquire the electrophysiological properties expected of fully developed neurons.

These results suggest that P19 cells may be used for studies related to neuronal cell development and maturation and that P19 cells may be considered for cell replacement strategies in neurodegenerative disorders of the central nervous system.

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Citation Excerpt :
The RA-differentiated P19 cells possess a number of properties characteristic for neurons in the mammalian nervous system, including a morphology that resembles the structure of cultured brain cells with small cell bodies and long axons and dendrites (McBurney et al., 1988; Yao et al., 1995), and they are post-mitotic as are neurons in the mammalian brain (McBurney et al., 1988). Pre- and post-synaptic morphological features of chemical synapses have been discovered in the RA-differentiated P19 cells (McBurney et al., 1988) and electrophysiological studies suggest that the synapses are functionally active (Morasutti et al., 1994; Magnuson et al., 1995). This model makes it possible rapidly to obtain a homogeneous and reproducible neural cell culture for screening studies.
The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity.
In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity.
A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells
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The P19 cell line neuronal-differentiation pathway, from neuroepithelial-like germinal cells to postmitotic neurons, closely resembles that occurring in the mammalian central nervous system (CNS) at both functional and morphological levels (Cheun and Yeh, 1991; Chiu et al., 1995; Finley et al., 1996; Jones-Villeneuve et al., 1982; MacPherson et al., 1997; MacPherson and McBurney, 1995; Parnas and Linial, 1997; Sharma and Notter, 1988; Tanaka et al., 1992). In addition, RA-induced P19 cells grafted into striata of adult rats mature to acquire electrophysiological properties of fully mature CNS neurons (Magnuson et al., 1995; Morassutti et al., 1994). The use of EC cell lines is restricted by functional limitations like in vitro approximation and heterogeneous-population in cell cultures, therefore making it difficult to address questions at the single cell-type level.
P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine β-d-arabinofuranoside and 2′-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro.
ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate
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Citation Excerpt :
Recently, Van den Berg et al., [39] identified ZFPIP/Zpf462 as a physical partner of Oct4, reinforcing the hypothetical link between these two proteins in a common molecular mechanism for maintaining pluripotency. As extensively reported in the literature, P19 cells treated with RA are known to differentiate into cells with “neuron-like” structures and functions [40–43]. They notably express neuronal marker genes such as Pax6, Mash-1 and Znf536 [18,22,23].
The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible.
We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells.
Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.
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Neurons were prepared for this study from P19 cells by retinoic acid induction via embryoid body formation (McBurney et al., 1982). P19 cells are an ideal model system for the de novo formation of neurons, which rapidly form without feeder cell layers and express neocortical or hippocampal markers (Bain et al., 1994; Finley et al., 1996; Magnuson et al., 1995; Morassutti et al., 1994; Staines et al., 1994). Embryoid bodies attached immediately to laminin-coated dishes but tenuously to dishes pretreated with poly-L-lysine or left untreated.
In neurons, the interaction of laminin with its receptor, β1 integrin, is accompanied by an increase in cytosolic Ca²⁺. Neuronal behavior is influenced by CaMK-II, the type II Ca²⁺/calmodulin-dependent protein kinase, which is enriched in axons of mouse embryonic neurons. In this study, we sought to determine whether CaMK-II is activated by laminin, and if so, how CaMK-II influences axonal growth and stability. Axons grew up to 200 μm within 1 day of plating P19 embryoid bodies on laminin-1 (EHS laminin). Activated CaMK-II was found enriched along the axon and in the growth cone as detected using a phospho-Thr²⁸⁷ specific CaMK-II antibody. β1 integrin was found in a similar pattern along the axon and in the growth cone. Direct inhibition of CaMK-II in 1-day-old neurons immediately froze growth cone dynamics, disorganized F-actin and ultimately led to axon retraction. Collapsed axonal remnants exhibited diminished phospho-CaMK-II levels. Treatment of 1-day neurons with a β1 integrin-blocking antibody (CD29) also reduced axon length and phospho-CaMK-II levels and, like CaMK-II inhibitors, decreased CaMK-II activation. Among several CaMK-II variants detected in these cultures, the 52-kDa δ variant preferentially associated with actin and β₃ tubulin as determined by reciprocal immunoprecipitation. Our findings indicate that persistent activation of δ CaMK-II by laminin stabilizes nascent embryonic axons through its influence on the actin cytoskeleton.
Poly(D,L lactic-co-glycolic acid) microspheres as biodegradable microcarriers for pluripotent stem cells
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The pluripotent nature and proliferative capacity of embryonic stem cells makes them an attractive cell source for tissue engineering and regeneration. In our study we investigated the use of poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres as biodegradable microcarriers of pluripotent cells and as delivery systems of bioactive factors, which influence cell differentiation. The pluripotent P19 embryonal carcinoma cell line was used as a model to study cell attachment, growth and differentiation of pluripotent stem cells on PLGA microspheres. Retinoic acid (RA) was encapsulated in the PLGA microcarriers to influence cell differentiation—more specifically, to induce P19 cell differentiation into neurons. The results revealed that P19 cells attach and grow on the surface of the RA loaded PLGA microspheres. Moreover, the RA loaded PLGA microspheres were shown to be as effective as soluble RA at inducing P19 cell differentiation into neurons. Hence, the results of these ex vivo studies clearly demonstrate the capacity of PLGA microspheres to serve a dual role as both delivery systems of bioactive factors and as scaffolds for pluripotent cells. More importantly, our study demonstrates the potential use of PLGA microspheres as transplantation matrices of pluripotent stem cells for tissue engineering and regeneration.
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Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum

Abstract

Gene

Devl Brain Res.

Devl Biol.

Trends Neurosci.

Trends Neurosci.

Prog. Brain Res.

Neuron

Neuroscience

Trends Neucosci.

J. Neurosci. Meth.

Neuroscience

Cell

Devl Biol.

Molec. Brain Res.

Cell

Trends Neurosci.

Brain Res. Rev.

Histochemical stains for macrophages in cell smears and tissue sections: β-galactosidase, acid phosphatase, nonspecific esterase, succinic dehydrogenase, and cytochrome oxidase

Induced muscle differentiation in an embryonal carcinoma cell line

Molec. Cell. Biol

Immortalization of bipotential and plastic glio-neuronal precursor cells

Transplantation of human fetal dopamine cells for Parkinson's disease: results at one year

Arch. Neurol.

Establishment of permanent astroglial cell lines, able to differentiate in vitro, from transgenic mice carrying the polyoma virus large T gene: an alternative approach to brain cell immortalization

J. Neurosci. Res.

Implantation of human fetal ventral mesencephalon into the right caudate nucleus in advanced Parkinson's disease

Arch. Neurol.