Method for artificial induction of differentiation of multipotential stem cells into neural precursor cells

Abstract

The invention discloses a method for artificial induction of differentiation of multipotential stem cells into neural precursor cells. The method comprises the following steps: performing in-vitro amplification culture on embryonic stem cells; performing subculture on the embryonic stem cells; and performing induction differentiation and in-vitro amplification on the embryonic stem cells into neural precursor cells. The invention discloses the method for artificial induction of differentiation of the multipotential stem cells into the neural precursor cells, in-vitro culture is implemented in a colony mode, peripheral cells can be relatively easily differentiated since cells in the middle of a colony are dense and the cells have relatively intense mutual action, meanwhile, donor cells used in the method are sufficient in source, high in purity and free of pollution, and thus a novel way is provided for acquiring nerve cells.

Description

technical field

[0001] The invention belongs to the field of biomedicine, in particular to a method for artificially inducing the differentiation of pluripotent stem cells into neural precursor cells. Background technique

[0002] Embryonic stem cells are derived from the inner cell mass of pre-implantation embryos, have self-renewal ability and developmental totipotency, and can differentiate into cell types representing the three germ layers in vivo and in vitro. Thomson established the first human embryonic stem cell line (hES cell ), making embryonic stem cells a big step forward from laboratory research to clinical application. Since then, research on hES cells has made rapid progress. Despite facing many ethical controversies, the application prospect of hES cells in future regenerative medicine is attracting more and more scientists. The in vitro differentiation of hES cells provides a good model for studying the cellular and molecular mechanisms of early embryonic d...

Examples

Embodiment 1

[0020] S1. Take human embryonic stem cells and inoculate them on BG02 medium, wherein the composition of the culture solution is: 15% KSR, 4ng / mL bFGF, 1.5mmol glutamine, 0.1mol / L β-mercaptoethanol, 1mmol / L non-essential amino acids, 50 μg / mL streptomycin, 50 μg / mL penicillin;

[0021] S2. The medium was placed at 37°C, 5% CO 2 Cultivate in an incubator, replace the culture medium every day, and after the cells are cultured to confluence, add 1mg / ml collagenase IV at 37°C for digestion and passage, inoculate on new feeder cells, the passage ratio is 1:5;

[0022] S3. Transfer the embryonic stem cells cultured in vitro to a four-well culture plate added with neural precursor cell basal medium, and digest them with 0.05% trypsin after 10 days of adherent culture;

[0023] The neural precursor cell basal medium is 50% NeuroBasalMedium, 1% N2 supplement, 1% B27, 1.5 mmol glutamine, 50 μg / mL streptomycin, 50 μg / mL penicillin added to DMEM / F12 medium;

[0024] S4. Inoculate the d...

Embodiment 2

[0026] S1. Human embryonic stem cells were inoculated on BG02 medium, wherein the composition of the culture medium was: 15% KSR, 4ng / mL bFGF, 2mmol glutamine, 0.15mol / Lβ-mercaptoethanol, 1mmol / L L non-essential amino acids, 50 μg / mL streptomycin, 50 μg / mL penicillin;

[0027] S2. The medium was placed at 37°C, 5% CO 2 Cultivate in an incubator, replace the culture medium every day, and after the cells are cultured to confluence, add 1mg / ml collagenase IV at 37°C for digestion and passage, inoculate on new feeder cells, the passage ratio is 1:6;

[0028] S3. Transfer the embryonic stem cells cultured in vitro to a four-well culture plate added with neural precursor cell basal medium, and digest them with 0.05% trypsin after 12 days of adherent culture;

[0029] The basal medium for neural precursor cells is 50% NeuroBasalMedium, 1% N2 supplement, 1% B27, 2 mmol glutamine, 50 μg / mL streptomycin, and 50 μg / mL penicillin added to DMEM / F12 medium;

[0030] S4. Inoculate the dige...

Embodiment 3

[0032] S1. Take human embryonic stem cells and inoculate them on BG02 medium, wherein the composition of the culture medium is: 15% KSR, 4ng / mL bFGF, 1.8mmol glutamine, 0.13mol / L β-mercaptoethanol, 1mmol / L non-essential amino acids, 50 μg / mL streptomycin, 50 μg / mL penicillin;

[0033] S2. The medium was placed at 37°C, 5% CO 2 Cultivate in an incubator, replace the culture medium every day, and after the cells are cultured to confluence, add 1 mg / ml collagenase IV at 37°C for digestion and passage, and inoculate on new feeder cells, the passage ratio is 1:5.5;

[0034] S3. Transfer the embryonic stem cells cultured in vitro to a four-well culture plate added with neural precursor cell basal medium, and digest them with 0.05% trypsin after 11 days of adherent culture;

[0035] The basal medium for neural precursor cells is 50% NeuroBasalMedium, 1% N2 supplement, 1% B27, 1.8 mmol glutamine, 50 μg / mL streptomycin, and 50 μg / mL penicillin added to DMEM / F12 medium;

[0036] S4. ...

technical field

[0001] The invention belongs to the field of biomedicine, in particular to a method for artificially inducing the differentiation of pluripotent stem cells into neural precursor cells. Background technique

[0002] Embryonic stem cells are derived from the inner cell mass of pre-implantation embryos, have self-renewal ability and developmental totipotency, and can differentiate into cell types representing the three germ layers in vivo and in vitro. Thomson established the first human embryonic stem cell line (hES cell ), making embryonic stem cells a big step forward from laboratory research to clinical application. Since then, research on hES cells has made rapid progress. Despite facing many ethical controversies, the application prospect of hES cells in future regenerative medicine is attracting more and more scientists. The in vitro differentiation of hES cells provides a good model for studying the cellular and molecular mechanisms of early embryonic d...

Examples

Embodiment 1

[0020] S1. Take human embryonic stem cells and inoculate them on BG02 medium, wherein the composition of the culture solution is: 15% KSR, 4ng / mL bFGF, 1.5mmol glutamine, 0.1mol / L β-mercaptoethanol, 1mmol / L non-essential amino acids, 50 μg / mL streptomycin, 50 μg / mL penicillin;

[0021] S2. The medium was placed at 37°C, 5% CO 2 Cultivate in an incubator, replace the culture medium every day, and after the cells are cultured to confluence, add 1mg / ml collagenase IV at 37°C for digestion and passage, inoculate on new feeder cells, the passage ratio is 1:5;

[0022] S3. Transfer the embryonic stem cells cultured in vitro to a four-well culture plate added with neural precursor cell basal medium, and digest them with 0.05% trypsin after 10 days of adherent culture;

[0023] The neural precursor cell basal medium is 50% NeuroBasalMedium, 1% N2 supplement, 1% B27, 1.5 mmol glutamine, 50 μg / mL streptomycin, 50 μg / mL penicillin added to DMEM / F12 medium;

[0024] S4. Inoculate the d...

Embodiment 2

[0026] S1. Human embryonic stem cells were inoculated on BG02 medium, wherein the composition of the culture medium was: 15% KSR, 4ng / mL bFGF, 2mmol glutamine, 0.15mol / Lβ-mercaptoethanol, 1mmol / L L non-essential amino acids, 50 μg / mL streptomycin, 50 μg / mL penicillin;

[0027] S2. The medium was placed at 37°C, 5% CO 2 Cultivate in an incubator, replace the culture medium every day, and after the cells are cultured to confluence, add 1mg / ml collagenase IV at 37°C for digestion and passage, inoculate on new feeder cells, the passage ratio is 1:6;

[0028] S3. Transfer the embryonic stem cells cultured in vitro to a four-well culture plate added with neural precursor cell basal medium, and digest them with 0.05% trypsin after 12 days of adherent culture;

[0029] The basal medium for neural precursor cells is 50% NeuroBasalMedium, 1% N2 supplement, 1% B27, 2 mmol glutamine, 50 μg / mL streptomycin, and 50 μg / mL penicillin added to DMEM / F12 medium;

[0030] S4. Inoculate the dige...

Embodiment 3

[0032] S1. Take human embryonic stem cells and inoculate them on BG02 medium, wherein the composition of the culture medium is: 15% KSR, 4ng / mL bFGF, 1.8mmol glutamine, 0.13mol / L β-mercaptoethanol, 1mmol / L non-essential amino acids, 50 μg / mL streptomycin, 50 μg / mL penicillin;

[0033] S2. The medium was placed at 37°C, 5% CO 2 Cultivate in an incubator, replace the culture medium every day, and after the cells are cultured to confluence, add 1 mg / ml collagenase IV at 37°C for digestion and passage, and inoculate on new feeder cells, the passage ratio is 1:5.5;

[0034] S3. Transfer the embryonic stem cells cultured in vitro to a four-well culture plate added with neural precursor cell basal medium, and digest them with 0.05% trypsin after 11 days of adherent culture;

[0035] The basal medium for neural precursor cells is 50% NeuroBasalMedium, 1% N2 supplement, 1% B27, 1.8 mmol glutamine, 50 μg / mL streptomycin, and 50 μg / mL penicillin added to DMEM / F12 medium;

[0036] S4. ...