CN103013917A - Method for inducing human amniotic mesenchymal stem cells to differentiate into neuron-like cells - Google Patents

Abstract

The invention provides a method for inducing differentiating human amniotic mesenchymal stem cells (hAMSCs) to differentiate into neuron-like cells by adopting all-trans retinoic acids, a basic fibroblast growth factor (bFGF) and an epidermal growth factor (EGF). The method comprises the following steps of: separating the hAMSCs, carrying out primary culture of the hAMSCs, subculturing and amplifying the hAMSCs, detecting hAMSCs immunophenotyping, inducing the hAMSCs to differentiate into the neuron-like cells and carrying out cellular immunity fluorescence staining. According to the inducing method provided by the invention, umbilical cord mesenchymal stem cells are induced to differentiate into neutral stem cells by using the all-trans retinoic acids in combination of the bFGF and the EGF; the neural stem cells not only have the typical morphology of nerve cells, but also express neuron marker antigen neuron-specific emolase and astrocyte marker antigen glial fibrillary acidic proteins; and the capability of a mesenchymal cell trans-germinal layer differentiating into non mesenchymal cells is realized so that the mesenchymal cells are likely to turn into more ideal seed cells for clinical application in further.

Description

Induction method for differentiating human amniotic mesenchymal stem cells into neuron-like cells

Technical Field

The invention relates to a method for inducing human amniotic mesenchymal stem cells (hAMSCs) to be differentiated into neuron-like cells in the technical field of biology, in particular to a method for inducing the human amniotic mesenchymal stem cells to be differentiated into the neuron-like cells by applying all-trans-retinoic acid, basic fibroblast growth factor and epidermal growth factor.

Background

The human amniotic mesenchymal stem cells (hAMSCs) maintain the plasticity of embryonic stem cells at the early stage of gastrulation and have larger multidirectional differentiation potential. hAMSCs can be differentiated into various tissue cells theoretically, and meanwhile, because amnion is convenient to obtain and avoids ethical disputes, the hAMSCs have the potential to become one of the stem cell sources for future clinical application. However, the neural cell transformation rate after the hAMSCs are transplanted is only 3% -10% in vivo, and the rate of differentiation into glial cells is high and the rate of differentiation into neuron-like cells is low under the in vivo environment, which undoubtedly brings adverse factors to the recovery of the nervous system function. If hAMSCs can be differentiated into neuron-like cells in vitro, the effect of cell transplantation can be achieved with half the effort.

Studies on the differentiation of mesoderm-derived mesenchymal stem cells (BMSCs) into ectoderm-derived neurons are in the beginning. All-trans retinoic acid is a vitamin A derivative, can obviously increase the number of nerve cells and has a dose-dependent effect, can regulate the differentiation of EGF (epidermal growth factor) responsive nerve stem cells, and increases the synthesis of neuronal cells and astrocytes. The compound is combined with neurotrophic factors and cytokines to induce embryonic stem cells to differentiate towards the neural stem cells, has similar development process in vivo and can partially simulate the in vivo environment, thereby being increasingly paid attention. However, these studies have mostly stayed on the level of neuronal morphological characteristics and expressed neuronal markers in post-induction hAMSCs, and lack evidence of physiological properties of neuronal cells, so many scholars believe that such cells are more appropriately termed "neuronal-like cells".

Disclosure of Invention

The invention provides a method for inducing human amniotic mesenchymal stem cells (hAMSCs) to differentiate into neuron-like cells, which adopts all-trans retinoic acid, alkaline fibroblast growth factor and epidermal growth factor to induce the human amniotic mesenchymal stem cells to differentiate into the neuron-like cells, not only has the typical morphology of nerve cells, but also expresses neuron-labeled antigen neuron specific enolase and astrocyte-labeled antigen glial fibrillary acidic protein, and specifically comprises the following steps: hAMSCs are separated, hAMSCs are primarily cultured, hAMSCs are subcultured and amplified, hAMSCs cell immunophenotype is detected, hAMSCs are induced to differentiate into neuron-like cells, and cell immunofluorescence staining is carried out.

Wherein the step of isolating the hAMSCs comprises: removing placenta from the placenta tissue by mechanical method under sterile condition, washing with D-Hanks solution, and cutting to obtain amnion with diameter of about 1mm³Adding 2.5g/L trypsin into the fragments, and digesting the fragments for 10 minutes at 37 ℃; adding DMEM containing 5% calf serum to stop digestion, gently blowing and beating, mixing uniformly, and filtering with a 200-mesh cell sieve; adding 1.0g/LII collagenase digestive juice into the filtered amniotic membrane tissue, and digesting for 0.5 hour at 37 ℃; adding DMEM containing 5% calf serum to stop digestion, gently blowing, beating, mixing uniformly, filtering with a 200-mesh cell sieve, and collecting cells; at 1000 rpmCentrifuging for 5 minutes under the condition; trypan blue staining was performed to count viable cells, and the cell density was adjusted to 1X 10⁶Inoculating to a 25cm2 flask, and adding DMEM medium containing 10ng/ml bFGF and 10% FBS; placing CO with the temperature of 37 ℃, the saturation humidity and the volume fraction of 5 percent₂Culturing in an incubator, observing the growth condition and morphological characteristics of primary and passage cells daily under an inverted phase contrast microscope, changing the liquid for 1 time every other day, and shooting and recording; after the confluence degree of the cells reaches 80%, digesting the cells for 2 to 3 minutes at 37 ℃ by using a 0.25% trypsin-0.02% EDTA (ethylene diamine tetraacetic acid) solution, adding a culture medium to stop the action of the trypsin, centrifuging the cells for 5 minutes at 1000 rpm, discarding supernatant, resuspending the cell sediment by using the culture medium, and then resuspending the cell sediment by using a 1 × 10 medium⁷Cell density passages per ml; in the process of passage, changing the liquid for 1 time every other day, and repeating the steps when the bottle bottom is full of 70% of cells;

the primary culture step of the hAMSCs comprises the following steps: transferring the amniotic cell suspension into a centrifuge tube, balancing, centrifuging for 15 minutes at 2000 rpm, removing supernatant, and collecting cells; blowing and beating the collected cells into uniform suspension by using 30mL of 0.01M PBS, centrifuging for 15 minutes at 1500 rpm, washing for 2 times, and collecting the cells; 3mL of FBS was aspirated before cell inoculation and T75cm was added²Type cell culture flask, put at 37 deg.C and 5% CO₂Incubating in a saturated humidity incubator for 30 minutes, and coating the culture bottle; discarding the coating solution, resuspending and digesting the obtained cells by using DMEM/F12 complete culture solution containing 20% FBS and 4ng/ml epidermal growth factor, uniformly blowing and beating the cells, and counting the cells; adjusting the cell concentration to 1.0X 10⁶Cells/ml, seeded on coated T75cm²Placing in a cell culture flask at 37 deg.C and 5% CO²Culturing in a cell culture box with saturated humidity, and dynamically observing by a phase contrast microscope; after culturing for 4-5 days, changing the liquid completely, discarding the non-adherent cells, and then changing the liquid half every 3-4 days; when the adherent growth of the cells is observed to reach 80-90% confluence, the cells are digested by 0.25% trypsin-0.01% EDTA, and the obtained cells are primary cells.

The steps of subculturing and amplifying hAMSCs comprise: when the adherent growth of the primary hAMSCs cells is observed to reach 80-90% confluence, the original culture in the culture bottle is discardedCulturing, sucking 10ml of 0.01M PBS buffer solution, slightly adding into a culture bottle for washing, and discarding the washing solution; adding 1.0ml of digestive juice containing 0.25% trypsin and 0.01% EDTA, covering the bottom of the bottle with a saturated solution, and observing under an inverted microscope; adding 1.0ml FBS to stop the digestion of pancreatin; adding 10ml of 0.01M PBS for repeated blowing and flushing, and centrifuging for 10 minutes at the room temperature of 900 rpm; discarding supernatant, adding 10ml DMEM/F12 to resuspend cells, and subculturing according to the proportion of 1: 2-1: 3; standing at 37 deg.C for 5% CO₂And culturing in a cell culture box with saturated humidity to obtain 1 st generation (P1) hAMSCs, and repeating the operations for culturing and amplifying P2, P3 and P4 … generation cells when the cells grow to 80-90% confluence.

The step of detecting the cellular immunophenotype of the hAMSCs comprises the following steps: the cultured cells of passage 2 or 3 were first digested with 0.25% trypsin and then adjusted to 1X 10 cell concentration with 1% bovine serum albumin in PBS⁷Per ml; the following murine anti-human monoclonal antibodies were added separately: CD34-FITC, CD45-PE, CD19-FITC, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC and HLA-DR-FITC, incubating at 4 ℃ for half an hour, washing with PBS for 1 time, and performing flow cytometry analysis.

The step of inducing the differentiation of the hAMSCs into neuron-like cells comprises the following steps: inoculating 2-3 generation cultured cells to 6-well plates respectively, and changing to an induction culture medium when the growth density reaches 60%; 37 ℃ and 5% CO₂And (5) carrying out static culture at saturated humidity.

The cellular immunofluorescent staining step comprises the following steps: cultured cells were stained with immunofluorescence 3 days after induced differentiation.

The invention uses all-trans-retinoic acid (ATRA), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) to induce human amniotic mesenchymal stem cells (hAMSCs) to differentiate into neuron-like cells, which not only have the typical form of nerve cells, but also express Neuron Specific Enolase (NSE) of neuron marker antigen and Glial Fibrillary Acidic Protein (GFAP) of astrocyte marker antigen; the BMSCs are suggested to still keep the capability of differentiating into non-mesenchymal cells by the trans-germ layer, can differentiate into neuron-like cells by the trans-germ layer, and are expected to become seed cells for nerve cell replacement therapy.

Drawings

The foregoing and other objects, features, and advantages of the invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings. Wherein:

fig. 1 is a flowchart illustrating steps of a method for inducing differentiation of human amniotic mesenchymal stem cells into neuron-like cells according to the present invention.

Detailed Description

Fig. 1 shows a flow chart of steps of the method for inducing differentiation of human amniotic mesenchymal stem cells into neuron-like cells, which is a method for inducing differentiation of human amniotic mesenchymal stem cells into neuron-like cells by using all-trans retinoic acid, basic fibroblast growth factor and epidermal growth factor, the method comprising the following steps:

s1, isolation of human amniotic mesenchymal stem cells (hAMSCs):

removing placenta from the placenta tissue by mechanical method, washing with D-Hanks solution for several times to remove residual blood stain, and cutting the washed amnion into about 1mm³Adding 2.5g/L trypsin into the fragments, and digesting the fragments for 10 minutes at 37 ℃; adding DMEM containing 5% calf serum to stop digestion, gently blowing and beating, mixing uniformly, and filtering with a 200-mesh cell sieve; adding 1.0g/L II type collagenase digestive juice into the filtered amniotic membrane tissue, and digesting for 0.5 hour at 37 ℃; adding DMEM containing 5% calf serum to stop digestion, gently blowing, beating, mixing uniformly, filtering with a 200-mesh cell sieve, and collecting cells; centrifuging for 5 minutes at 1000 rpm; trypan blue staining was performed to count viable cells, and the cell density was adjusted to 1X 10⁶Perml, inoculated to 25cm²The culture flask is added with 10ng/mlDMEM medium with bFGF and 10% FBS; placing CO with the temperature of 37 ℃, the saturation humidity and the volume fraction of 5 percent₂Culturing in an incubator, observing the growth condition and morphological characteristics of primary and passage cells daily under an inverted phase contrast microscope, changing the liquid for 1 time every other day, and shooting and recording; after the confluence degree of the cells reaches 80%, digesting the cells for 2 to 3 minutes at 37 ℃ by using a 0.25% trypsin-0.02% EDTA (ethylene diamine tetraacetic acid) solution, adding a culture medium to stop the action of the trypsin, centrifuging the cells for 5 minutes at 1000 rpm, discarding supernatant, resuspending the cell sediment by using the culture medium, and then resuspending the cell sediment by using a 1 × 10 medium⁷Cell density passages per ml; during the passage, the liquid is changed every other day for 1 time, and the steps are repeated when the bottle bottom is full of 70 percent of cells.

S2, primary culture of hAMSCs:

transferring the amniotic cell suspension into a centrifuge tube, balancing, centrifuging for 15 minutes at 2000 rpm, removing supernatant, and collecting cells; blowing and beating the collected cells into uniform suspension by using 30mL of 0.01M PBS, centrifuging for 15 minutes at 1500 rpm, washing for 2 times, and collecting the cells; 3mL of FBS was aspirated before cell inoculation and T75cm was added²Type cell culture flask, put at 37 deg.C and 5% CO₂Incubating in a saturated humidity incubator for 30 minutes, and coating the culture bottle; discarding the coating solution, resuspending and digesting the obtained cells by using DMEM/F12 complete culture solution containing 20% FBS and 4ng/ml Epidermal Growth Factor (EGF), uniformly blowing and beating the cells, and counting the cells; adjusting the cell concentration to 1.0X 10⁶Cells/ml, seeded on coated T75cm²Placing in a cell culture flask at 37 deg.C and 5% CO²Culturing in a cell culture box with saturated humidity, and dynamically observing by a phase contrast microscope; after culturing for 4-5 days, changing the liquid completely, discarding the non-adherent cells, and then changing the liquid half every 3-4 days; when the adherent growth of the cells is observed to reach 80-90% confluence, the cells are digested by 0.25% trypsin-0.01% EDTA, and the obtained cells are primary cells.

S3, subculturing and amplifying hAMSCs:

observing the adherent growth of the primary hAMSCs cells until 80-90% confluence, removing the original culture solution in the culture bottle, sucking 10ml of 0.01M PBS buffer solution, and slightly adding the buffer solution into the culture bottleInternal washing, and removing washing liquid; adding 1.0ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of the bottle with a soaked solution, observing under an inverted microscope, wherein the cell clearance is increased, the cytoplasm is retracted, and shaking and blowing the bottle to ensure that the cells are circularly floated; adding 1.0ml FBS to stop the digestion of pancreatin; adding 10ml of 0.01M PBS for repeated blowing and flushing, and centrifuging for 10 minutes at the room temperature of 900 rpm; discarding supernatant, adding 10ml DMEM/F12 for resuspension of cells, and subculturing according to the ratio of 1: 2-1: 3; the culture system is 20% FBS, DMEM/F12 complete culture solution 15 ml/bottle of 4ng/ml EGF. Standing at 37 deg.C for 5% CO₂And culturing in a cell culture box with saturated humidity, counting as 1 st generation (passage l, P1) hAMSCs, and repeating the operations to perform P2, P3 and P4 … generation cell culture amplification when the cells grow to 80-90% confluence.

S4, detection of hAMSCs cellular immunophenotype:

the cultured cells of passage 2 or 3 were first digested with 0.25% trypsin and then adjusted to 1X 10 cell concentration with 1% bovine serum albumin in PBS⁷Per ml; the following murine anti-human monoclonal antibodies were added separately: CD34-FITC, CD45-PE, CD19-FITC, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC and HLA-DR-FITC, incubating for half an hour at 4 ℃, washing for 1 time by PBS, and performing flow cytometry detection and analysis; flow cytometry assays showed that BMSCs expressed CD29, CD44, and CD 106.

S5, hAMSCs induced differentiation into neuron-like cells:

inoculating 2-3 generation cultured cells to a 6-well plate respectively, and changing to an induction culture medium (DMEM + 10% FBS +1 mu mol/L ATRA +20ng/ml bFGF +20ng/ml EGF) when the growth density reaches 60%; 37 ℃ and 5% CO₂Carrying out static culture at saturated humidity; after the inducing liquid is added for 2 hours, the cell morphology is obviously changed, and the cytoplasm of the BMSCs shrinks towards the nucleus under a light mirror to be in a typical perikaryoid morphology; most cells can form a neuron-like cell shape after 3-5 hours, the cell body is circular, the protrusion is long, branches appear at the tail end of the protrusion, the protrusions of part of adjacent cells are connected into a net, and the number of the cells is not obviously increased; after 3 days most cells were converted to bipolarOr a multipolar neuronal cell-like morphology, extending synapses (like axons or dendrites) with a network of partial cells drawn between them.

S6, cell immunofluorescence staining:

cultured cells were stained with immunofluorescence 3 days after induced differentiation. Adherent cells are removed from a culture medium, washed 3 times by PBS, fixed by 4% paraformaldehyde for 30 minutes, washed 3 times by PBS, treated for 20 minutes by 0.3% TritonX-100, rinsed 3 times by PBS, sealed for 20 minutes at room temperature by 10% sheep serum, aspirated, added with rabbit anti-human NSE (1: 500) and GFAP (1: 1000), incubated for 2 hours at 37 ℃, washed 3 times by PBS, added with goat anti-mouse-FITC (1: 500) and incubated for 30 minutes, and stained to 50-70% NSE, 25-50% GFAP positive and Nestin positive cells are reduced to 1.6% at 48 hours.

The invention uses all-trans-retinoic acid (ATRA), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) to induce human amniotic mesenchymal stem cells (human amnion membrane mesenchymal stem cells, hAMSCs) to be differentiated into neuron-like cells, not only has the typical morphology of nerve cells, but also expresses nerve cell marker proteins, and prompts that BMSCs still retain the capability of differentiating into non-mesenchymal cells by spanning germ layers, can differentiate into neuron-like cells by spanning germ layers, and is expected to become seed cells for nerve cell replacement therapy.

The present invention is not limited to the embodiments described above, and those skilled in the art may make modifications or changes within the scope of the disclosure without departing from the spirit of the present invention, so that the scope of the present invention is defined by the appended claims.

Claims (2)

1. A method for inducing differentiation of human amniotic mesenchymal stem cells into neuron-like cells is characterized in that the method applies all-trans-retinoic acid combined with bFGF and EGF to induce the differentiation of umbilical mesenchymal stem cells into neural stem cells, and comprises the following steps: hAMSCs separation, hAMSCs primary culture, hAMSCs subculture and amplification, hAMSCs cell immunophenotype detection, hAMSCs induced differentiation into neuron-like cells and cell immunofluorescence staining; wherein,

the step of isolating the hAMSCs comprises: collecting fresh placenta after delivery under aseptic condition, and adoptingMechanically peeling amnion from placenta tissue, washing with D-Hanks solution, and cutting the washed amnion into about 1mm³Adding 2.5g/L trypsin into the fragments, and digesting the fragments for 10 minutes at 37 ℃; adding DMEM containing 5% calf serum to stop digestion, gently blowing and beating, mixing uniformly, and filtering with a 200-mesh cell sieve; adding 1.0g/L II type collagenase digestive juice into the filtered amniotic membrane tissue, and digesting for 0.5 hour at 37 ℃; adding DMEM containing 5% calf serum to stop digestion, gently blowing, beating, mixing uniformly, filtering with a 200-mesh cell sieve, and collecting cells; centrifuging for 5 minutes at 1000 rpm; trypan blue staining was performed to count viable cells, and the cell density was adjusted to 1X 10⁶Perml, inoculated to 25cm²Adding a DMEM medium containing 10ng/ml bFGF and 10% FBS into the culture flask; placing CO with the temperature of 37 ℃, the saturation humidity and the volume fraction of 5 percent₂Culturing in an incubator, observing the growth condition and morphological characteristics of primary and passage cells daily under an inverted phase contrast microscope, changing the liquid for 1 time every other day, and shooting and recording; after the confluence degree of the cells reaches 80%, digesting the cells for 2 to 3 minutes at 37 ℃ by using a 0.25% trypsin-0.02% EDTA (ethylene diamine tetraacetic acid) solution, adding a culture medium to stop the action of the trypsin, centrifuging the cells for 5 minutes at 1000 rpm, discarding supernatant, resuspending the cell sediment by using the culture medium, and then resuspending the cell sediment by using a 1 × 10 medium⁷Cell density passages per ml; in the process of passage, changing the liquid for 1 time every other day, and repeating the steps when the bottle bottom is full of 70% of cells;

the primary culture step of the hAMSCs comprises the following steps: transferring the amniotic cell suspension into a centrifuge tube, balancing, centrifuging for 15 minutes at 2000 rpm, removing supernatant, and collecting cells; blowing and beating the collected cells into uniform suspension by using 30mL of 0.01M PBS, centrifuging for 15 minutes at 1500 rpm, washing for 2 times, and collecting the cells; 3mL of FBS was aspirated before cell inoculation and T75cm was added²Type cell culture flask, put at 37 deg.C and 5% CO₂Incubating in a saturated humidity incubator for 30 minutes, and coating the culture bottle; discarding the coating solution, resuspending and digesting the obtained cells by using DMEM/F12 complete culture solution containing 20% FBS and 4ng/ml epidermal growth factor, uniformly blowing and beating the cells, and counting the cells; adjusting the cell concentration to 1.0X 10⁶Cells/ml, seeded on coated T75cm²Placing in a cell culture flask at 37 deg.C and 5% CO²Saturation ofCulturing in a cell culture box with humidity, and dynamically observing by a phase contrast microscope; after culturing for 4-5 days, changing the liquid completely, discarding the non-adherent cells, and then changing the liquid half every 3-4 days; when the adherent growth of the cells is observed to reach 80-90% confluence, the cells are digested by 0.25% trypsin-0.01% EDTA, and the obtained cells are primary cells;

the steps of subculturing and amplifying hAMSCs comprise: when the adherent growth of the primary hAMSCs cells is observed to reach 80-90% confluence, absorbing the original culture solution in the culture bottle, absorbing 10ml of 0.01M PBS buffer solution, slightly adding the buffer solution into the culture bottle for washing, and removing the washing solution; adding 1.0ml of digestive juice containing 0.25% trypsin and 0.01% EDTA, covering the bottom of the bottle with a saturated solution, and observing under an inverted microscope; adding 1.0ml FBS to stop the digestion of pancreatin; adding 10ml of 0.01M PBS for repeated blowing and flushing, and centrifuging for 10 minutes at the room temperature of 900 rpm; discarding supernatant, adding 10ml DMEM/F12 for resuspension of cells, and subculturing according to the ratio of 1: 2-1: 3; standing at 37 deg.C for 5% CO₂Culturing in a cell culture box with saturated humidity to obtain 1 st generation (P1) hAMSCs, and repeating the above operations to perform P2, P3 and P4 … generation cell culture amplification when the cells grow to 80-90% confluence;

the step of detecting the cellular immunophenotype of the hAMSCs comprises the following steps: the cultured cells of passage 2 or 3 were first digested with 0.25% trypsin and then adjusted to 1X 10 cell concentration with 1% bovine serum albumin in PBS⁷Per ml; the following murine anti-human monoclonal antibodies were added separately: CD34-FITC, CD45-PE, CD19-FITC, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC and HLA-DR-FITC, incubating for half an hour at 4 ℃, washing for 1 time by PBS, and performing flow cytometry detection and analysis;

the step of inducing the differentiation of the hAMSCs into neuron-like cells comprises the following steps: inoculating 2-3 generation cultured cells to 6-well plates respectively, and changing to an induction culture medium when the growth density reaches 60%; 37 ℃ and 5% CO₂Carrying out static culture at saturated humidity; and

the cellular immunofluorescent staining step comprises the following steps: cultured cells were stained with immunofluorescence 3 days after induced differentiation.

2. The method for inducing differentiation of human amniotic mesenchymal stem cells into neuron-like cells according to claim 1, wherein the induction medium in the step of inducing differentiation of hAMSCs into neuron-like cells is DMEM + 10% FBS + 1. mu. mol/LATRA +20ng/ml bFGF +20ng/ml EGF.

Images