Stem cells from the mammalian neural plate

Abstract

The present invention relates to methods for deriving novel stem cells from the mammalian early neural plate.

Claims

1. An in vitro method of obtaining and maintaining isolated neural plate stem cells (NPSC), comprising: (a) providing pluripotent cells or primitive neurectodermal cells in dissociated form; (b) culturing the cells in the presence of FGF4 for a sufficient amount of time to obtain an isolated NPSC by detecting the expression of the marker Brn-2 and not detecting the expression of the markers Ngn2, MASH1, Pax3, Pax6, En1, En2, and Krox20 on the NPSC, and (c) maintaining the isolated NPSC in culture in the presence of FGF4, wherein the NPSCs maintain the expression of Brn-2 and do not express Ngn2, MASH1; Pax3; Pax6; En1; En2; and Krox20.

2. The method of claim 1 wherein the pluripotent cells or primitive neurectodermal cell is a primary embryonic cell obtained from the early neural plate.

3. The method of claim 1 wherein the pluripotent cell is an epiblast stem cell, an embryonic stem cell, or an induced pluripotent cell.

4. The method of claim 3 wherein the pluripotent cell is cultured in the presence of FGF2 and/or Activin in addition to FGF4.

5. The method of claim 1 wherein the pluripotent cell or primitive neurectodermal cell is cultured in the presence of FGF4 and in the absence of FGF2 and/or Activin.

6. The method of claim 5 wherein the pluripotent cell or primitive neurectodermal cell is cultured in serum free medium containing exogenous FGF4.

7. The method of claim 1, wherein the isolated NPSC is maintained in culture for at least 40 passages.

8. The method of claim 1, wherein the NPSC are cultured in the presence of an extracellular matrix.

9. The method of claim 8, wherein the extracellular matrix comprises one or more component selected from the group consisting of fibronectin, laminin and gelatin.

10. An in vitro method of differentiating NPSCs into dopaminergic or serotonergic neurons, the method comprising: (a) obtaining NPSCs by the method of claim 1, (b) culturing the NPSCs in the presence of Shh and FGF8 in the absence of FGF4, (c) detecting tyrosine hydroxylase on the cultured cells, wherein serotonergic neurons that express tyrosine hydroxylase are produced.

11. An in vitro method of differentiating NPSCs into motor neurons, the method comprising (a) obtaining NPSCs by the method of claim 1, and (b) culturing the NPSCs in the presence of retinoic acid and in the absence of FGF4, and (c) detecting HB-9 on the cultured cells, wherein motor neurons expressing HB-9 are produced.

12. An in vitro method of maintaining a population of isolated neural plate stem cells (NPSCs) expressing Brn-2 in an undifferentiated state, comprising: (a) detecting Brn-2 but not Ngn2, MASH1; Pax3; En1; En2; and Krox20 on the population of isolated neural plate stem cells, and (b) culturing the NPSCs in neuronal medium comprising FGF4, wherein the NPSCs maintain expression of Brn-2 and do not express Ngn2, MASH1; Pax3; En1; En2; and Krox20.

13. The method of claim 12, wherein the neuronal medium is serum free medium.

14. The method of claim 12, wherein the neuronal medium is N2 medium.

15. The method of claim 12, wherein the NPSCs are derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) by the steps of: culturing the iPSCs or ESCs cells in neuronal medium comprising FGF4 for a sufficient amount of time to differentiate the iPSCs or ESCs to NPSCs, and detecting expression of Brn-2 but not Ngn2, MASH1; Pax3; Pax6; En1; En2; and Krox20 on the NPSCs.

16. The method of claim 12, wherein the culturing step is in neuronal medium in the absence of FGF2 and activin.

17. The method of claim 12, wherein the NPSCs are derived from epiblast stem cells, wherein the NPSCs are derived by a method comprising: (a) culturing the epiblast stem cells in the presence of fibronectin, FGF2, Activin and FGF4 for a sufficient amount of time to form colonies with neural plate morphology, (b) subsequently culturing the cells of (a) in medium comprising FGF4 in the absence of additional factors to obtain NPSCs, and (c) detecting expression of Brn-2 and not Ngn2, MASH1; Pax3; Pax6; En1; En2; and Krox20 on the NPSCs.

18. The method of claim 12, wherein the FGF4 is present in an amount of at least 50 ng/ml.

Description

EXAMPLES

(1) The invention is described in specific embodiments with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the effects of FGFs on Proliferation and Survival of E7.5 CNS Progenitors

(3) Legend: Cells were dissociated in 0.5% trypsin and plated into N2+FGF4+Trypsin Inhibitor (2 mg/ml, from Chicken Egg white [Sigma, T-2011]).

(4) N2 media change+FGF4 at 24 hours. At 48 hours, N2 media change+specified FGF. After 1 hour, BrdU added. Cells sit for an additional hour, then fixed.

(5) FIG. 2 shows the In vitro Culture of E8.5 Neural Stem Cells

(6) Legend: E8.5 dissociated ventral midbrain cultured in the presence of Shh (500 ng/ml) and FGF8 (100 ng/ml). Cells were photographed 5 days after plating.

(7) FIG. 3 shows the production of NPSCs from pluripotent cells

(8) Legend:

(9) Step 1. Pluripotent cell conditions (FGF2+Activin for epiblast cells)+FGF4.

(10) Step 2. Cells look like epiblast cells with minor differences in morphology.

(11) Step 3. After 4-6 passages (12-18 days), Colonies with neural plate morphology emerge. FGF2 and Activin are removed.

(12) Cells are serially passaged with Accutase in N2+FGF4 or N2+FGF4+BMP4 (up to 41 passages, so far). Alternatively, passaging cells with Collagenase preserves the neural plate morphology of the primary cells.

(13) FIG. 4 shows methods for producing NPSCs from epiblast stem cells

(14) Legend: Overgrowth of mouse epiblast cells. All growth factors are removed at 50% confluence. Medium added every 2-3 days, but is not completely changed.

(15) There is not very much cell death. After 7 days many clusters of neural plate cells can be observed and expanded with FGF4. In the absence of FGF4, a subpopulation of different cells are observed (the identity of these non-neural cells is not known at this time).

(16) Alternatively, Epiblast cells can be grown under non-adherent conditions in N2+FGF4. Within 48-72 hours, aggregates are observed. The aggregate cultures are supplemented with fibronectin daily. After 3-4 days the aggregates adhere and begin to spread. Neural plate stem cells are observed.

(17) FIG. 5 shows that FGF4 suppresses the Expression of Neurogenic Transcription Factors

(18) Legend: MASH1 polyclonal antibody (1:1,000) generously provided by J. Johnson.

(19) Ngn2 monoclonal antibody (1:50) generously provided by D. J. Anderson.

(20) Sox1 polyclonal antibody (1:200) generously provided by R. Lovell-Badge.

(21) PLZF polyclonal antibody (1:50) obtained from Calbiochem.

(22) FIG. 6 shows the derivation of Neural Progenitors from Human ES cells

(23) Legend: (Top three panels) The appearance of neural plate cells derived from human embryonic stem cells. Note the neuroepithelial nature of the colonies and the density of the cells. (Graph) Just like primary mouse neural plate cells, neural plate cells derived from human embryonic stem cells proliferate specifically in response to FGF4 (compare to FIG. 1).

(24) FIG. 7 shows that FGF4 suppresses the Morphogenic Response of Shh and BMP2

(25) Legend: Nkx2.2 (T. Jessell) and Pax3 (C. Ordahl) monoclonal antibodies from DSHB. Used at 1:20. BMP+FGF4 still has a pro-survival effect. The neural plate cells don't exhibit patterning characteristic of cells derived from the later embryo. FGF4 suppresses the effect of sonic hedgehog (Shh) and BMP2. However in the absence of FGF4 the cells further differentiate and the expected morphogenic response to Shh and BMP2 is seen.

(26) FIG. 8 shows the differentiation of NPSCs to Neurons in the absence of FGF4

(27) Legend: 18 days growth factor (FGF4) withdrawal (p+19). Tuj1=monoclonal antibody binding neuronal beta-tubulin; TH=tyrosine hydroxylase. 7 days growth factor (FGF4) withdrawal (p+23) at low density Many undifferentiated cells can still be observed

(28) FIG. 9 shows the induction of Dorsoventral Identities by Morphogens in the Absence of FGF4

(29) Legend: Msx1 (T. Jessell) monoclonal antibody from DSHB. Used at 1:20. Nkx6.1 polyclonal antibody (1:400) generously provided by M. German. This data shows the Neural plate stem cells do not, for example, express the dorsal marker Pax3 when cultured in the presence of FGF4. However, Pax3 expression is observed when FGF4 is withdrawn.

(30) FIG. 10 shows that treatment with Shh leads to an Increase in Monoaminergic Neurons

(31) Legend: TH polyclonal antibody (Pel-freez, 1:400). Pitx3 polyclonal antibody (Zymed, 1:400) 5-HT rabbit polyclonal antibody (Sigma, 1:4,000). Lmx1b gp polyclonal antibody (1:4,000) generously provided by T. Jessell.

(32) FIG. 11 shows that Retinoic Acid-Treated Progenitors Differentiate to Putative Motor Neurons

(33) Legend: Tuj1 polyclonal antibody (Covance, 1:400). Hb9 (T. Jessell) monoclonal antibody from DSHB. Used at 1:20.

(34) FIG. 12 shows floor plate induction of neural plate cells. Increasing doses of Shh are able to induce Foxa2, a marker of the floor plate, in neural plate stem cells. Inhibition of MAPK signaling is able to enhance the percentage of Foxa2 induction by Shh. Floor plate cells give rise to dopamine neurons.

(35) FIG. 13 shows derivation of Neural Plate Stem Cells on a variety of extracellular matrices in monolayer culture

(36) Legend: Human induced pluripotent cell (hIPS) line, KIPS, were grown by standard protocols in human embryonic stem cell medium plus FGF2 and activin on mouse embryonic feeders (MEFs). At passage 26, hIPS colonies were passaged using collagenase. Colonies were separated from MEFs by gravity in a 15 mL conical tube containing hES medium. Cells were subsequently replated into 12 well-plates containing N2 medium plus FGF4 (100 ng/mL) and the well plates were coated either with fibronectin, laminin, or gelatin. The fibronectin and laminin-coated plates were pre-coated with polyornithine. After four days, the cells were fixed in cold 4% paraformaldehyde and stained by immunofluorescence for the neural plate marker, Brn2, and the apoptosis marker, cleaved caspase-3.

(37) The results show that Neural Plate Stem Cells (NPSCs) are successfully derived on laminin and gelatin-coated plates.

(38) FIG. 14 shows clonal growth of neural plate stem cells

(39) Legend: Single neural plate neural stem cells were plated at low density and grown into clonally derived neural plate stem cell colonies (one colony is shown). Individual colonies were selected and expanded to form clonal neural plate stem cell lines. So far, the clonal neural plate stem cell lines which have been tested are all able to give rise to neurons, including dopamine and serotonin neurons.

(40) FIGS. 15 & 15A show expression of Sox1 and Brn2 in the Neural Plate

(41) Legend: Sox1 has been believed to be the earliest marker of the neural lineage. However, the inventors have found that Sox1 is not a good marker for neural plate stem cells and that, in fact, Sox1 is actually not a good marker for the early neural plate in vivo. The figures shown illustrate the expression of Sox1 and Brn2 in the early embryo by immunohistochemistry. (FIG. 15) Sox1 is not expressed in the early neural plate. Sox1 is observed to be restricted to the ventral midline at intermediate neural plate stages. (FIG. 15A(A)) Sox1 is first seen to be expressed in precisely two rows of cells at the midline. (FIG. 15A(B)) Unlike Sox1, Brn2 is widely expressed throughout the neural plate at comparable stages.

(42) FIG. 16 shows NPSC transplantation to a Chick Embryo

(43) Legend: Images of neural plate cells injected into the neural plate of a chicken embryo. The chicken embryos were left for several days and sacrificed at midgestation. In both images, it is clear that the neural plate cells have differentiated into neurons. (A) GFP-labeled mouse neural plate cell-derived neuron. (B) Human neural plate-derived neuron stained with a human nuclear antigen. The neuron is also tyrosine hydroxylase-positive (TH) which is an enzyme involved in dopamine synthesis, a catecholamine centrally involved in Parkinson's disease.

(44) FIG. 17 shows NPSC transplantation into Neonatal Rat Hindbrain

(45) Legend: Neural plate cells injected into neonatal rat cortex. (A) survival of mouse antigen specific cells, stained with a mouse-specific antibody at the edge of the graft (mouse-negative cells at the bottom of the image are easily accounted). (B) & (C) Grafts of human cells into rat brains. (B) Numerous Tuj1+ young+ neurons and GFAP+ glia within the graft. (C) Several TH+ putative, dopamine neurons can be seen, as well as one serotonergic (5-HT) cell.

MATERIALS AND METHODS

(46) The composition of the N2 medium described herein is as set out in Bottenstein and Sato. 1979.

Example 1

(47) Derivation of Neural Plate Stem Cells from the Mouse Embryo

(48) Mouse headfold-stage embryos (E7.5-E7.75) were removed from the uteri of timed, pregnant mothers. The anterior neural plate was dissected away from the visceral endoderm, head mesenchyme, and the developing foregut and heart primordium. Cells were dissociated and placed in N2 medium on fibronectin-coated dishes.

(49) Unlike rosette-forming cells taken from later-staged embryos or derived from pluripotent cells (Elkabetz et al. 2008; Koch et al. 2009), the neural plate stem cells form colonies of flat, continuous epithelium. The neural plate stem cells, like embryonic stem cells, have large, prominent nuclei and a high nucleus-to-cytoplasm ratio. Sox1-expressing cells are present and the non-neural markers, Oct4, brachyury, and Sox17, are all absent.

(50) At various, later stages, FGF2 and EGF have been shown to support the multipotent state and proliferation of neural stem cells (Cattaneo and McKay 1990; Pollard 2008). In contrast, neither FGF2 nor EGF maintained the neural plate stem cells in their undifferentiated state. Either in the presence or absence of FGF2, neural plate stem cells derived from mouse embryos begin to form rosette structures (Elkabetz et al. 2008, Koch et al. 2009) within 48 hours; these rosettes differentiate to form neurons within one week.

(51) We screened a number of fibroblast growth factors and found, to our surprise, that FGF4 has a potent and specific mitogenic effect on neural plate stem cells that is not mimicked by other FGFs. In addition, as measured by immunohistochemistry for cleaved caspase-3, FGF4 promotes the survival of neural plate stem cells.

Example 2

(52) Derivation of Neural Plate Stem Cells from Mouse and Human Pluripotent Cells

(53) We sought to derive neural plate stem cells from mouse pluripotent cells. Mouse epiblast cells were maintained in N2 medium on fibronectin in the presence of FGF2 and activin. Under these conditions, cultures were supplemented with FGF4.

(54) In separate experiments, neural plate stem cells have been derived directly from human pluripotent cells (hEs and iPS) through culture in N2 medium supplemented with only FGF4 (i.e. in the absence of FGF2, activin or any other growth factor).

(55) Aside from the mitogenic properties of FGF4 on neural plate stem cells, FGF4 has also been implicated in the differentiation of pluripotent cells (Kunath 2007, Stavridis et al. 2007) and FGFs are believed to have a role in neural induction (Stern; Pera et al. 2003).

(56) Initially, the epiblast stem cells appear polarized upon FGF4 treatment. It is not clear if the development of a bipolar morphology is indicative of a transition in cell state to ectoderm, or if this is merely a change in cell shape.

(57) After 4-6 passages (roughly 12-18 days), colonies with a neural plate morphology appear, at which time FGF2 and activin are removed. Continued application of FGF2 and activin, at this stage, leads to the development of homogeneous non-neural cells; the identity of these non-neural cells is not known at this time.

(58) The resulting pluripotent cell-derived neural plate stem cells are grown on fibronectin in N2 medium plus FGF4 and passaged using accutase.

(59) Neural plate stem cells can be derived from mouse epiblast stem cells in N2 plus FGF4, in the absence of FGF2 and activin. In this case, colonies with neural plate morphology can be observed more rapidly (approximately one week) but this is accompanied by massive cell death in the cultures.

(60) Like neural plate stem cells derived from the embryo, the pluripotent-cell derived NPSC's do not express markers of non-neural lineages and, when FGF4 is removed, they form rosettes en route to neuronal differentiation. Differentiation to neurons has been observed from neural plate stem cells passaged up to 41 times.

(61) The key feature of the methods of deriving NPSC's is the step of culturing the precursor cells (epiblast stem cells, ES, iPS, primary embryonic) with FGF4. Some example methods comprising this step for deriving NPSC's from epiblast stem cells are outlined in FIG. 4.

(62) Sox1 is believed to be the earliest marker of the definitive neural lineage in the vertebrate (Pevny 1998). While Sox1 expression is observed by immunohistochemistry in neural plate stem cells, it is only seen in about 30% of the cells and there is some variation in the intensity of nuclear staining. We find that when FGF4 is removed, Sox1 is expressed in nearly all of the resulting rosette cells at 48 hours.

(63) In addition, the neurogenic bHLH transcription factors, MASH1 and Ngn2 (Parras 2002) which are not expressed in the neural plate, are also induced after 48 hours of FGF4 withdrawal. Finally, in a similar vein, the neural rosette marker, PLZF, is upregulated after FGF4 withdrawal. These results indicate that Sox1 may not be a distinctive marker for the early neural plate.

(64) We investigated Sox1 expression in the mouse neural plate by immunohistochemistry. To our surprise, Sox1 is not expressed in the neural plate when it is initially formed; instead, Sox1 is first seen at intermediate neural plate stages when it is restricted to the two rows of hinge-forming cells at the ventral midline of the neural plate which initiate neural tube closure (Smith J L 1991). Even at later stages, sox1 is expressed weakly, if at all, in the lateral neural plate (Thomas Andreska, R. K., and A. G. S, in preparation). In contrast, the POU-domain and homeodomain-containing transcription factor, Brn2/Pou3f2 is expressed throughout the neural plate from the earliest stages and is uniformly expressed in vitro in neural plate stem cells.

Example 3

(65) Patterning of Neural Plate Stem Cells.

(66) In order to specifically drive the differentiation of neural progenitors, they should be responsive to secreted factors which pattern the neural tube. For the dorsoventral axis of the neural tube, the floor plate and roof plate organizers secrete sonic hedgehog (shh) and bone morphogenetic proteins which have ventralizing and dorsalizing activities, respectively.

(67) Markers of dorsoventral identity are not expressed in the open neural plate (Shimamura 1997; Liem 1995) but are induced at approximately E8.25 in the closed or almost closed neural tube. Similarly, neural plate stem cells do not express markers of dorsoventral identity in the presence of FGF4 and merely adding Shh or BMP2 is not sufficient to induce Nkx2.2, a ventral marker, or Pax3, a dorsal marker, respectively. If FGF4 is withdrawn, just as Sox1 and bHLH factors are induced, dorsoventral markers are similarly upregulated and, under these conditions, Shh induces Nkx2.2 and BMP2 induces Pax3. FGF4 seems to maintain neural plate stem cells in an unpatterned state, a state before they acquire the competence to be patterned by Shh or BMP-2.

(68) Morphogens like Shh act in a concentration-dependent manner. In a larger experiment, we grew neural plate stem cells, withdrawn from FGF4, in three different concentrations of Shh (0 ng/ml, 500 ng/ml, and 1 ug/ml) and also in BMP-2 and assessed the expression of four dorsoventral markers.

(69) As the concentration of Shh is increased, ventral markers (Nkx2.2 and Nkx6.1) are induced at the expense of the dorsal marker (Pax3); upon BMP2 treatment, the opposite effect is observed. In addition, under these conditions, the roof plate marker, Msx1, is only induced upon BMP2 treatment. Similar observations have been made in the spinal cord and midbrain of the intact embryo (Briscoe 2000; Agarwala 2001), in primary neural tube explants (Wijgerde 2002) and dissociated progenitors (Kittappa 2007), and in differentiating mouse embryonic stem cells (Wichterle 2002. Neural plate stem cells, released from the effects of FGF4, are fully capable of responding to patterning by ventralizing and dorsalizing morphogens.

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