JP2005179257A - Novel embryonic stem cell differentiation inducer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a novel embryonic stem cell differentiation inducer which induces the differentiation of embryonic stem cells, for example, the differentiation of mouse embryo-derived tumor cells, P19 cells, into nerve-like cells. <P>SOLUTION: Carotenoids induce the differentiation of, for example, mouse embryo-derived tumor cells, P19 cells, into nerve-like cells. The carotenoids are exemplified by lutein and β-carotene. Carotenoids are contained in an extract prepared by extracting with e.g., ethanol a culture obtained by cultivating microbes belonging to the genus Stenotrophomonas or the genus Fravobacterium, and these carotenoids also induce the differentiation of stem cells into nerve-like cells. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel embryonic stem cell differentiation inducer capable of differentiating, for example, mouse embryo-derived tumor cells P19 cells into nerve-like cells.

P19 cells are mouse embryo-derived tumor cell lines that can differentiate into various cells derived from ectoderm, mesoderm, and endoderm. It has been reported that retinoic acid differentiates these cells into neurons, astrocytes, and fibroblast-like cells (MacPherson and McBurney, 1995).
The nerves differentiated from the cultured cells are irreversibly terminated, and in the form of typical neurons, there are many central nervous system cell markers: neuron-specific enolase, neurofilament protein, neuro-specific nuclear antibody ( NeuN) has been confirmed to have the characteristics of central neurons.
Most studies on in vitro differentiation of mouse P19 cells have been performed using retinoic acid (MacPherson et al. 1997, Parnas and Linial 1995, Dedhar et al. 1991).

The search for new chemical substances that induce differentiation has been eagerly conducted by many researchers all over the world to investigate the mechanism of cell differentiation. However, retinoic acid has developed P19 cells into fully developed neurons. The exact mechanism of differentiation is still unclear.
Embryonic stem cells are used as models for early human embryogenesis, development tools for new growth factors and drugs, and have potential as a supply cell for transplantation medicine.
Therefore, there are high demands and high expectations for new chemical substances that induce differentiation into various cells.

MacPherson P.A and McBurney M.W. (1995) P19 embryonal carcinoma cells: A source of cultured neurons amenable to genetic manipulation.Methods, A Companion to Methods in Enzymology 7: 238-252. MacPherson P.A., Jones, S., Pawson P.A., Marshall K.C. and McBurney M.W. (1997). P19 cells differentiate in to glutamatergic and glutamate-responsive neurons in vitro. Neuroscience, 80: 487-499. Parnas, D. and Linial M. (1995) Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19). International Journal of Developmental Neuroscience, 13: 767-781. Dedhar S., Robertson, K and Gray V. (1991) Induction of expression of the αvβ1 and αvβ3 Integrin heterodimers during retinoic acid-induced neuronal differentiation of Murine embryonal carcinoma cells. Journal of Biological Chemistry, 266: 21846-21852

  An object of the present invention is to provide a novel embryonic stem cell differentiation inducer capable of differentiating embryonic stem cells.

The present invention has been made by finding that carotenoids induce differentiation of mouse embryo-derived tumor P19 cells into nerve-like cells, for example, as a result of extensive studies.
Examples of carotenoids include lutein and β-carotene.
In addition, various carotenoids are contained in an extract obtained by extracting a cultured microorganism of the genus Stenotrophomonas or Flavobacterium with ethanol etc., and these carotenoids also cause the stem cells to be neuronal. It became clear that differentiation was induced in cells.

When these carotenoids were added to 1 μM culture medium and cultured for a predetermined period, nerve-like cells with long neurites formed from both ends were observed.
Morphological observations such as the shape, size, neurofilament length, and branching pattern of neuronal-like cells induced by carotenoids induce differentiation by retinoic acid (a substance commonly known as a differentiation inducer). Consistent with the neuronal morphology observed.
Furthermore, staining was carried out using an anti-mouse neuronal nucleus monoclonal antibody (NeuN), a secondary antibody in which an anti-mouse neurofilament 160 clone NN18 marker antibody and fluorescein were bound, and the characteristics of the nerve-like cells induced by carotenoids were confirmed.
These results presented in this study suggest that carotenoids are candidates for new chemicals that differentiate P19 cells into neuronal cells.
Furthermore, the results of this study indicate that P19 cells possess a receptor that recognizes the carotenoids examined this time, and in the future, a new promoter that promotes differentiation of embryonic stem cells into nerves and other cells It is expected to be a clue to elucidate factors.

  In the present invention, it is suggested that carotenoids may be novel inducers for differentiating embryonic stem cells, which could be confirmed by immunocytochemical examination of differentiated cells.

(P19 cell culture)
Mouse embryonic tumor cells P19 cells were cultured according to a conventional method. That is, undifferentiated P19 cells are a medium in which α-mineral essential medium (α-MEM, GIBCO BRL) is added with 10% fetal bovine serum, 0.36% (w / v) sodium bicarbonate, and 1% antibiotic mixture. The cells were cultured at 37 ° C. in a 5% CO 2 incubator. The culture was maintained so as to grow exponentially in 5 × 10 ⁷ monolayers per 10 ml.
(Induction of stem cell differentiation)
1 × 10 ⁶ cells collected by trypsin treatment are suspended in 3-5 ml of culture solution (α-MEM supplemented with 2-8% fetal calf serum) so as not to adhere to the culture dish. They were plated on a culture dish (35 mm diameter) pre-coated with bovine serum albumin.
Here, a positive control (retinoic acid), a standard carotenoid (lutein, β-carotene), and a microorganism-derived carotenoid extract were added to each culture dish to a final concentration of 0.1 to 10 μM and cultured. In any case, cell aggregates are formed in one day of culture (Fig. 1).
After culturing for 4 days, cells were collected by trypsin treatment and re-cultured on a cover glass coated with an adhesive such as poly-L-lysine. Cells were obtained.

(Separation and purification of carotenoid extract)
The carotenoid extract was extracted from a cultured cell of Stenotrophomonas genus D1 (Accession No. FERM P-18481) and Flavobacterium genus P104 (Accession No. FERM BP-5006) with a mixed solvent of acetone and hexane, The solvent was concentrated by distillation under reduced pressure, and it was adjusted by dissolving in 100% ethanol.
As a result of HPLC analysis, lutein, canthaxanthin, astaxanthin, and β-carotene were detected as major carotenoids from D1 of the genus Stenotrophomonas.
From P104 of the genus Flavobacterium, zeaxanthin, β-cryptoxanthin, echinenone and β-carotene were detected as main carotenoids.

(Immunocytochemistry experiment of neuron-like cells derived from P19 cells)
In immunocytochemistry experiments of neuron-like cells derived from P19 cells obtained by treatment with carotenoids or retinoic acid, a mouse anti-neuronal cell nuclear monoclonal antibody (NeuN, Chemicon, USA) was used as a primary antibody to stain the nucleus. A mouse anti-neurofilament 160 clone NN18 marker antibody (Sigma, USA) was used as the primary antibody for staining the neurofilament, and a fluorescein-conjugated secondary antibody (Chemicon, USA) was used as the secondary antibody.
The differentiated cells were fixed by incubation for 10 minutes at room temperature in a phosphate buffer containing paraformaldehyde according to a standard method.
The cells were washed with phosphate buffer and blocked by incubation in phosphate buffer containing 0.5% (w / v) bovine serum albumin at 37 ° C. for 30 minutes (operation to prevent nonspecific binding of antibody). .
100-fold diluted NeuN antibody or anti-neurofilament 160 clone NN18 marker antibody was added and incubated overnight at 4 ° C.
After washing with phosphate buffer, the fluorescein-conjugated secondary antibody was incubated at 37 ° C. for 4 hours and stained immunochemically.

(result)
Mouse embryo-derived tumor 19 cells did not spontaneously differentiate by continuing culture, but differentiated into neuronal cells by application of lutein, β-carotene, and carotenoid extract.
The differentiation process starting from the formation of cell aggregates was almost the same as that induced by retinoic acid (FIG. 1).
In addition, when cells plated on a cover glass coated with poly-L-lysine were cultured in a serum-free medium, it was observed that a structure extending from both ends of the cell body appeared after 24 hours.
The size and shape of the cell bodies and neurofilaments were confirmed to grow longer the next day. The shape of the nucleus and the length of the neurofilament of nerve-like cells differentiated by carotenoid treatment were the same as those of neurons differentiated by retinoic acid as a positive control (FIG. 2).
The cultured cells to which the carotenoid extract from the cultured microorganisms was applied differentiated more into nerve-like cells than when the standard concentrations of standard carotenoids lutein and β-carotene were applied, forming a pseudo-neural network.
Moreover, the neurofilaments of nerve-like cells were thicker when lutein or β-carotene was applied than when retinoic acid or carotenoid extract was applied.

It is confirmed by immunocytochemistry using NeuN that is a neuron nucleus specific marker and anti-neurofilament 160 clone NN18 marker antibody that cells that are thought to be morphologically neuron-like cells have the characteristics of nerve cells. Confirmed (FIG. 3).
NeuN clearly stained cell nucleus, and NN18 marker antibody clearly stained neurofilament.
Staining with NN18 marker antibody confirmed the possibility of multiple types of neuronal cells.
From the results of this study, it was shown that carotenoids are novel differentiation-inducing substances (substances capable of differentiating P19 cells) that were clearly unknown so far.

An example of a P19 cell aggregate is shown. A is not added, and B is a carotenoid extract derived from the genus Stenotrophomonas. Aggregates similar to B were observed by addition of either retinoic acid or carotenoids. 2 shows neuronal-like cell differentiation of P19 cells. A is P19 cells cultured without addition of differentiation inducer, B is retinoic acid added, C is lutein added, D is β-carotene added. Even when the carotenoid extract was added, cell differentiation similar to B-C was observed. An example of the immunochemical staining result is shown. A is obtained by staining cells differentiated by lutein with an anti-neuronal cell nuclear antibody (NeuN), and the cell nucleus is specifically stained. B is a cell obtained by staining a cell differentiated with a carotenoid extract derived from the genus Stenotrophomonas with an anti-neurofilament 160 clone NN18 marker, and the filament is specifically stained. Similar staining results were obtained with these specific antibodies using any differentiation inducer used in the present invention.

Claims (5)

  Stem cell differentiation inducer consisting of carotenoids.   2. The differentiation inducer according to claim 1, wherein the stem cell is an embryonic stem cell.   The differentiation-inducing substance according to claim 1 or 2, wherein the carotenoid is lutein or β-carotene.   The differentiation-inducing substance according to claim 1 or 2, wherein the carotenoids are extracts from cultured microorganisms.   5. The differentiation inducer according to claim 4, wherein the cultured microorganism is a genus Stenotrophomonas or a genus Flavobacterium.