METHOD FOR INDUCING DIFFERENTIATION OF FUNCTIONAL CEREBRAL CORTEX CELLS

Abstract

The present disclosure provides a medium and a method for inducing differentiation into functional cerebral cortical cells, wherein the medium comprises a neural medium and a nutritional supplement and the nutritional supplement is selected from a group consisting of SU5402, BIBF1120, IBMX and glucose. The method describes that specific factors such as an inhibitor of FGF signaling pathway, an inhibitor of VEGF signaling pathway and/or an activator of cAMP are added at specific time points during the induced differentiation process to accelerate the differentiation and maturation of the neural cells. Said method can produce stable and healthy neural cells with major functions at about 7 to 14 days after the initiation of the induced differentiation starting from human neural progenitor cells, with reduced manufacturing cost and shortened production time.

Claims

1. A medium for inducing differentiation into functional cerebral cortical cells, comprising a neural medium and a nutritional supplement, wherein the nutritional supplement comprises an inhibitor of FGF/VEGF signaling pathway and an activator of cAMP, wherein the inhibitor of FGF/VEGF signaling pathway is SU5402 and/or, BIBF1120, and wherein the activator of cAMP is IBMX alone, or a combination of IBMX and glucose.

2. The medium according to claim 1, wherein the nutritional supplement comprises BIBF1120, IBMX and glucose.

3. The medium according to claim 1, wherein the nutritional supplement comprises 80 nM˜100 μM SU5402, 1˜500 ng/mL BIBF1120, 1˜100 μM IBMX and 1˜10 mM glucose.

4. A method for inducing differentiation into functional cerebral cortical cells, comprising: neural stem cells or neural progenitor cells are inoculated on a cell culture plate after being dissociated, cultured in Neural Differentiation Medium A from day 1, and then cultured in Neural Differentiation Medium B from day 7; wherein the Neural Differentiation Medium A comprises one or more of retinoic acid, BDNF, GDNF, ascorbic acid, nutritional supplement, Neurobasal medium, and B-27 Supplement Minus Vitamin A; the Neural Differentiation Medium B comprises one or more of BDNF, GDNF, ascorbic acid, a nutritional supplement, Neurobasal medium, and B-27 Supplement Minus Vitamin A; and the nutritional supplement comprises an inhibitor of FGF/VEGF signaling pathway and an activator of cAMP, wherein the inhibitor of FGF/VEGF signaling pathway is SU5402 and/or BIBF1120, wherein the activator of cAMP is IBMX alone, or a combination of IBMX and glucose.

5. The method according to claim 4, wherein the nutritional supplement comprises SU5402, BIBF1120, IBMX and glucose.

6. The method according to claim 4, wherein the nutritional supplement comprises 80 nM˜10 μM SU5402, 1˜500 ng/mL BIBF1120, 1˜100 μM IBMX and 1˜10 mM glucose.

7. The method according to claim 6, wherein the nutritional supplement comprises 100 nM SU5402, 200 ng/mL BIBF1120, 10 μM IBMX and 5 mM glucose.

8. The method according to claim 4, wherein the Neural Differentiation Medium A comprises 2 μM retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/mL BIBF1120, 10 μM IBMX, 5 mM glucose, the Neurobasal medium and the B-27 Supplement Minus Vitamin A, wherein the ratio of the Neurobasal medium to the B-27 Supplement Minus Vitamin A is 50:1; and the Neural Differentiation Medium B comprises 20 ng/mL BDNF, 20 ng/mL GDNF 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/mL BIBF1120, 10 μM IBMX, 5 mM glucose, the Neurobasal medium and the B-27 Supplement Minus Vitamin A, wherein the ratio of the Neurobasal medium to the B-27 Supplement Minus Vitamin A is 50:1.

9. The method for according to claim 8, wherein culturing is conducted in the Neural Differentiation Medium A from day 1, with half of the medium being replaced every 3 to 5 days; culturing is continued by using Neural Differentiation Medium B starting from day 7, with half of the medium being exchanged every 3 to 5 days.

10. The method according to claim 9, wherein the neural stem cells or neural progenitor cells are dissociated by accutase and plated on poly-D-lysine/laminin-coated plate at a density of 5×10.sup.5/cm.sup.2.

11. The method according to claim 6, wherein the nutritional supplement comprises 80 nM˜10 μM SU5402, 150˜250 ng/mL BIBF1120, 5˜15 μM IBMX and 3˜8 mM glucose.

12. The method according to claim 6, wherein the nutritional supplement comprises 10 μM SU5402.

13. The method according to claim 6, wherein the nutritional supplement comprises 5 μM SU5402 and 50 μM IBMX.

14. The method according to claim 6, wherein the nutritional supplement comprises 5 ng/mL BIBF1120 and 50 μM IBMX.

Description

DESCRIPTION OF THE DRAWINGS

[0029] In order to illustrate the embodiments of the present disclosure or the technical solutions of the prior art more clearly, the drawings as needed for the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description only relates to examples of the present disclosure. Other drawings can also be contemplated by a person having ordinary skills in the art from the provided drawings of the present disclosure without any creative efforts.

[0030] FIG. 1 is the schematic flow diagram of the method provided in the present disclosure for inducing differentiation into functional cerebral cortical cells;

[0031] FIG. 2 shows the bright-field photos of human neural cells on day 1, 7, 14, and 84 of the induced differentiation of Example 1;

[0032] FIG. 3 shows examples of action potentials of human neural cells recorded by single-cell patch clamp, examples of miniIPSC (inhibitory neural firing) and miniEPSC (excitatory neural firing) on day 14 of the induced differentiation of Example 1;

[0033] FIG. 4 shows the statistical data of the amplitude and frequency of the excitatory and inhibitory neural firing of the cells, which were recorded on day 14 of the induced differentiation of Example 1;

[0034] FIG. 5 shows an exemplified record of sEPSC (spontaneous excitatory neural firing) of human neural cells recorded by single cell patch clamp on day 14 of the induced differentiation;

[0035] FIG. 6 shows an exemplified record of spontaneous neural firing of human neural cells on day 14 of the induced differentiation of Example 1;

[0036] FIG. 7 shows the results of immunofluorescence staining of MAP2 on day 14 of the differentiation;

[0037] FIG. 8 is a histogram showing the results of immunofluorescence staining of MAP2 on day 14 of the differentiation;

[0038] FIG. 9 shows the results of immunofluorescence staining of Synapsin on day 14 of the differentiation;

[0039] FIG. 10 is a histogram showing the results of immunofluorescence staining of Synapsin on day 14 of the differentiation;

[0040] FIG. 11 shows the results of immunofluorescence staining of vGlut on day 21 of the differentiation;

[0041] FIG. 12 is a histogram showing the results of immunofluorescence staining of vGlut on day 21 of the differentiation;

[0042] FIG. 13 is the bright-field photos of human neural cells on day 21 of the differentiation of Example 2 and Comparative Example 1;

[0043] FIG. 14 is a histogram showing the statistical results of dispersion degree of human neural cells on day 21 of the differentiation of Example 2 and Comparative Example 1;

[0044] FIG. 15 is a histogram showing the results of immunofluorescence staining of Synapsin on day 21 of the differentiation of Example 2 and Comparative Example 1;

[0045] FIG. 16 is a histogram showing the results of immunofluorescence staining of FOXG1 on day 21 of the differentiation of Example 2 and Comparative Example 1;

[0046] FIG. 17 is a statistic graph showing the record of spontaneous neural firing of human neural cells on day 7 of the differentiation of Example 2 and Comparative Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS

[0047] The technical solutions in the examples of the present disclosure will be described explicitly and comprehensively hereinafter. Obviously, only a part of the embodiments of the present disclosure is described herein. All other embodiments obtainable by one with ordinary skills in the art based on the examples of the present disclosure without making any creative efforts are within the protection scope of the present disclosure.

EXAMPLE 1

[0048] Step 1: The neural progenitor cells (hNPCs) generated by differentiation from human induced pluripotent stem cell line DYR0100 were dissociated by accutase, and then plated on poly-D-lysine/laminin-coated plate at a density of 5×10.sup.5/cm.sup.2.

[0049] Step 2: Starting from day 1, culturing was conducted by using Neural Differentiation Medium A at 37° C., 5% CO.sub.2 in the cell culture incubator for 7 days, with half of the medium being replaced every 3 days. The Neural Differentiation Medium A comprised in final concentrations of 2 μM retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/mL BIBF 1120, 10 μM IBMX, and 5 mM glucose in the Neurobasal medium and the B-27 Supplement Minus Vitamin A, wherein the ratio of the Neurobasal medium to B-27 Supplement Minus Vitamin A was 50:1.

[0050] Step 3: Starting from day 7, the culturing was conducted by using Neural Differentiation Medium B at 37° C., 5% CO.sub.2 in the cell culture incubator, with half of the medium being replaced every 3 days. The Neural Differentiation Medium B comprised in final concentrations of 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/mL BIBF1120, 10 μM IBMX, and 5 mM glucose in the Neurobasal medium and the B-27 Supplement Minus Vitamin A, wherein the ratio of Neurobasal medium to B-27 Supplement Minus Vitamin A was 50:1. Starting from day 7, the maturity-level of the neural cells could be measured by methods such as electrophysiological and immunofluorescence staining, and became ready to use when the predetermined functional index was realized.

[0051] Referring to FIG. 1, which is a schematic flow diagram of the method provided by the present disclosure for inducing differentiation into functional cerebral cortical cells, hNSCs or hNPCs were subjected to induced differentiation after being dissociated; the Neural Differentiation Medium A was used from starting day 1 of the induced differentiation; the Neural Differentiation Medium B was used starting from day 7; basic neural functions and maturation markers became detectable from day 14; and human cerebral cortical neural cells could be generated after further maturation. Specifically, the Neural Differentiation Medium A comprised in final concentrations of 2 μM retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/mL BIBF1120, 10 μM IBMX, 5 mM glucose in the Neurobasal and B-27 (Minus Vitamin A) medium; the Neural Differentiation Medium B comprised in final concentrations of 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 100 nM SU5402, 200 ng/mL BIBF1120, 10 μM IBMX, and 5 mM glucose, in the Neurobasal and B-27 (Minus Vitamin A) medium.

[0052] Referring to FIG. 2, FIG. 3 and FIG. 4, FIG. 2 shows the bright-field photos of human neural cells on day 1, 7, 14, and 84 of the induced differentiation of Example 1; FIG. 3 shows examples of action potentials of human neural cells recorded by single-cell patch clamp, examples of mIPSCs (inhibitory neural firing) and mEPSCs (excitatory neural firing) on day 14 of the induced differentiation of Example 1; FIG. 4 shows the statistical data of the amplitude and frequency of the excitatory and inhibitory firing of the cells, which were recorded on day 14 of the induced differentiation of Example 1. As shown in FIG. 2, FIG. 3 and FIG. 4, the neural cells including excitatory and inhibitory neurons with electrophysiological functions and could be subjected to long-term culturing became obtainable on day 14 by using the method of the present disclosure.

COMPARATIVE EXAMPLE 1

[0053] Different from Example 1, a common neural medium (Neurobasal medium+B-27) was used instead of the Neural Differentiation Medium A and Neural Differentiation Medium B.

COMPARATIVE EXAMPLE 2

[0054] Different from Example 1, a medium promoting the neurophysiological maturity of neural cells (BrainPhys) manufactured by a Canadian company well-known for stem cell reagents (CompA) was used instead of the Neural Differentiation Medium A and Neural Differentiation Medium B.

COMPARATIVE EXAMPLE 3

[0055] Different from Example 1, a medium supplement promoting the maturity of neural cells by BrainXell (CompB), a company run by a well-known American professor in the field of stem cell neural differentiation, was used instead of the Neural Differentiation Medium A and Neural Differentiation Medium B.

[0056] Referring to FIG. 5 and FIG. 6, FIG. 5 shows an exemplified record of sEPSC (spontaneous excitatory neural firing) of human neural cells recorded by single cell patch clamp on day 14 of the induced differentiation, in which Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1 are shown from top to bottom sequentially. FIG. 6 shows an exemplified record of spontaneous neural firing of human neural cells on day 14 of the induced differentiation of Example 1 (recorded by MEA (Multi Electrode Array), Axion Bioscience). It can be seen that the association of clustered action potential firings between multiple electrodes was found through software analysis. This represents that on day 14 certain network connections (a representation of a more mature function) were formed by the neural cells generated by the differentiation method provided in Example 1. However, the same phenomenon was not observed on neural cells generated by other differentiation methods (Day 14). Also, according to the reported literatures, such a phenomenon can hardly be observed on any human neural cells generated by culturing with other differentiation methods or media even in 2 months of differentiation.

[0057] Referring to FIG. 7 and FIG. 8, FIG. 7 shows the results of immunofluorescence staining of MAP2 on day 14 of the differentiation of Example 1 and Comparative Examples 1˜3, of which Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1 are shown from left to right, respectively; and bright-field photos, DAPI staining results, and MAP2 staining results are shown from top to bottom, respectively. FIG. 8 is a histogram showing the results of immunofluorescence staining of MAP2 on day 14 of the differentiation of Comparative examples 1˜3 and Example 1, of which Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1 are shown from left to right.

[0058] Referring to FIG. 9 and FIG. 10, FIG. 9 shows the results of immunofluorescence staining of Synapsin on day 14 of the differentiation of Comparative Examples 1˜3 and Example 1, of which Comparative Example 1, Comparative Example 3, and Example 1 are shown from left to right, respectively, and the Merged results, MAP2 staining results and Synapsin staining results are shown from top to bottom, respectively. FIG. 10 is a histogram showing the results of immunofluorescence staining of Synapsin on day 14 of the differentiation of Example 1 and Comparative Examples 1˜3, of which Comparative Example 1, Comparative Example 3, and Example 1 are shown from left to right, respectively.

[0059] Referring to FIG. 11 and FIG. 12, FIG. 11 shows the results of immunofluorescence staining of vGlut on day 21 of the differentiation of Example 1 and Comparative Examples 1˜3, of which Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1 are shown from left to right, respectively, and the Merge results, DAPI staining images and vGlut staining images are shown from top to bottom, respectively. FIG. 12 a histogram showing the results of immunofluorescence staining of vGlut on day 21 of the differentiation of Example 1 and Comparative Examples 1˜3, of which Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1 are shown from left to right, respectively.

[0060] It can be known from FIG. 7˜FIG. 12 that neural cells including excitatory and inhibitory neurons can be fast generated by the method provided in the present disclosure.

EXAMPLE 2

[0061] Step 1: the neural progenitor cells (hNPCs) generated by differentiation from human induced pluripotent stem cell line DYR0100 were dissociated by accutase, and then plated on a poly-D-lysine/laminin-coated plate at a density of 5×10.sup.5/cm.sup.2.

[0062] Step 2: Starting from day 1, culturing was conducted by using Neural Differentiation Medium A at 37° C., 5% CO.sub.2 in the cell culture incubator for 7 days, with half of the medium being replaced every 3 days. The Neural Differentiation Medium A comprised in final concentrations of 2 μM retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 10 μM SU5402 in Neurobasal medium and B-27 Supplement Minus Vitamin A, wherein the ratio of the Neurobasal medium to B-27 Supplement was 50:1.

[0063] Step 3: Starting from day 7, the culturing was conducted by using Neural Differentiation Medium B at 37° C., 5% CO.sub.2 in the cell culture incubator, with half of the medium being replaced every 3 days. The Neural differentiation medium B comprised in final concentrations of 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 10 μM SU5402 in the Neurobasal medium and B-27 Supplement (Minus Vitamin A), wherein the ratio of the Neurobasal medium to the B-27 Supplement was 50:1. Starting from day 7, the maturity-level of the neural cells could be measured by methods such as electrophysiological and immunofluorescence staining, and became ready to use when the predetermined functional index was realized.

[0064] Referring to FIG. 13˜FIG. 14, FIG. 13 is the bright-field images of the human neural cells on day 21 of the induced differentiation of Example 2 and Comparative Example 1; FIG. 14 is the statistical results of dispersion degree of the cells on day 21 of the induced differentiation of Example 2 and Comparative Example 1.

[0065] Referring to FIG. 15˜FIG. 16, FIG. 15 is a histogram showing the results of immunofluorescence staining of Synapsin on day 21 of the differentiation of Example 2 and Comparative Example 1.

[0066] FIG. 16 is histogram showing the results of immunofluorescence staining of FOXG1 on day 21 of the differentiation of Example 2 and Comparative Example 1.

[0067] Referring to FIG. 17, FIG. 17 is the statistic results of the spontaneous neural firing records of the human neural cells on day 7 of the differentiation in Example 2 and Comparative Example 1 (recorded by MEA, Axion Bioscience). No electrophysiological signal was detected on day 7 for the cells in the Neural medium.

[0068] It can be known from FIG. 13˜FIG. 17, the method provided by the present disclosure can inhibit cell aggregation and facilitate maturation of neurons, such that the neural cells are sufficiently spaced, which is favorable for the growth of neurites and for further maturation into functional neurons.

EXAMPLE 3

[0069] Step 1: The neural progenitor cells (hNPCs) generated by differentiation from human induced pluripotent stem cell line DYR0100 were dissociated by accutase, and then plated on a poly-D-lysine/laminin-coated plate at a density of 5×10.sup.5/cm.sup.2.

[0070] Step 2: Starting from day 1, culturing was conducted by using Neural Differentiation Medium A at 37° C., 5% CO.sub.2 in the cell culture incubator for 7 days, with half of the medium being replaced every 3 days. The Neural Differentiation Medium A comprised in final concentrations of 2 μM retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 5 μM SU5402, and 50 μM IBMX in Neurobasal medium and B-27 Supplement Minus Vitamin A, wherein the ratio of the Neurobasal medium to B-27 Supplement was 50:1.

[0071] Step 3: Starting from day 7, the culturing was conducted by using Neural Differentiation Medium B at 37° C., 5% CO.sub.2 in the cell culture incubator, with half of the medium being replaced every 3 days. The Neural differentiation medium B comprised in final concentrations of 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 5 μM SU5402 and 50 μM IBMX in Neurobasal medium and B-27 Supplement (Minus Vitamin A), wherein the ratio of the Neurobasal medium to the B-27 Supplement was 50:1. The results showed that the method provided in Example 3 could effectively inhibit cell aggregation in comparison with Comparative Example 1, Comparative Example 2, and Comparative Example 3.

EXAMPLE 4

[0072] Step 1: The neural progenitor cells (hNPC) generated by differentiation from human induced pluripotent stem cell line DYR0100 were dissociated by accutase, and then plated on a poly-D-lysine/laminin-coated plate at a density of 5×10.sup.5/cm.sup.2.

[0073] Step 2: Starting from day 1, culturing was conducted by using Neural Differentiation Medium A at 37° C., 5% CO.sub.2 in the cell culture incubator for 7 days, with half of the medium being replaced every 3 days. The Neural Differentiation Medium A comprised in final concentrations of 2 μM retinoic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 5 ng/mL BIBF1120, and 50 μM IBMX in Neurobasal medium and B-27 Supplement Minus Vitamin A, wherein the ratio of the Neurobasal medium to the B-27 Supplement was 50:1.

[0074] Step 3: Starting from day 7, the culturing was conducted by using Neural Differentiation Medium B at 37° C., 5% CO.sub.2 in the cell culture incubator, with half of the medium being replaced every 3 days. The Neural differentiation medium B comprised in final concentrations of 20 ng/mL BDNF, 20 ng/mL GDNF, 0.2 mM ascorbic acid, 5 ng/mL BIBF1120, and 50 μM IBMX in Neurobasal medium and B-27 Supplement (Minus Vitamin A), wherein the ratio of the Neurobasal medium to B-27 Supplement was 50:1. The results showed that the method provided in Example 3 could effectively inhibit cell aggregation in comparison with Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0075] The above described examples are only preferred embodiments of the present invention. It should be pointed out that for persons with ordinary skills in the art, improvements and embellishments can be made without departing from the sprits of the present disclosure, and these improvements and embellishments shall also be regarded as within the scope of protection of the present disclosure.