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

Human amnion mesenchymal stem cell is divided into the induction method of neuron cell

Technical field

A kind of human amnion mesenchymal stem cell (hAMSCs) that the present invention relates to biological technical field is divided into the neuron cell induction method, and particularly a kind of all-trans-retinoic acid, Prostatropin, Urogastron used induces human amnion mesenchymal stem cell to be divided into the method for neuron cell.

Background technology

Human amnion mesenchymal stem cell (human amnion membrane mesenchymal stemcells, hAMSCs) has been kept the plasticity-of gastrula formation pre-embryo stem cell, has larger multi-lineage potential.HAMSCs can be divided into various histocytes in theory, and the while obtains convenient because of amnion and avoided ethics dispute, one of potential source of human stem cell that becomes following clinical application.But the Neural differentiation rate only was 3%～10% in vivo after hAMSCs transplanted, and the ratio that is divided in vivo neurogliocyte under the environment is large and to be divided into the ratio of neuron cell less, and this bring unfavorable factor can for undoubtedly the recovery of nervous function.If can be neuron cell in vitro differentiation with hAMSCs, the Transplanted cells of going again has the effect of getting twice the result with half the effort.

The mesenchymal stem cells MSCs (BMSCs) in mesoderm source is in the starting stage to the research of the neuron differentiation in ectoderm source.All-trans-retinoic acid is the derivative of vitamin A, can obviously increase the quantity of neurocyte and be dose-dependent effect, can regulate the cell differentiation of nerve cord of EGF (Urogastron) reaction, increases the synthetic of neuronal cell and astroglia cell.Itself and neurotrophic factor and cytokine are united for inducing embryo stem cell neuralward stem cell direction differentiation, and growth course is similar in the consubstantiality, and can the partial simulation internal milieu, therefore day by day comes into one's own.But, these researchs rest on more induces rear hAMSCs to have on neuron morphology feature and the expression neurocyte marker level, and lack the evidence with neurocyte physiology characteristic, therefore many scholars to think that this class cell is called as " neuron cell " more appropriate.

Summary of the invention

The invention provides a kind of human amnion mesenchymal stem cell (hAMSCs) and be divided into the neuron cell induction method, it is to use all-trans-retinoic acid, Prostatropin, Urogastron induces human amnion mesenchymal stem cell to be divided into neuron cell, the representative configuration that has not only had neurocyte, and expression neurone sign antigen neuronspecific enolase, star spongiocyte sign antigen glial fibrillary acidic protein, described method specifically may further comprise the steps: hAMSCs separates, the former culture of hAMSCs, going down to posterity of hAMSCs cultivated and amplification, the detection of hAMSCs Immunophenotyping, hAMSCs is induced to differentiate into neuron cell and immunofluorescent staining.

Wherein, the step that described hAMSCs separates comprises: get the fresh human placenta of throwing aside postpartum under the aseptic condition, adopt mechanical process that amnion is peeled off from placenta tissue, with the flushing of D-Hanks liquid, the amnion after the rinsing is cut into approximately 1mm with eye scissors ³Fragment adds 37 ℃ of digestion of 2.5g/L trypsinase 10 minutes; Add the DMEM contain 5% calf serum and stop digestion, softly blow and beat behind the mixing with the filtration of 200 order cells sieve; Add 1.0g/LII Collagenase Type Digestive system in the amnion tissue after the filtration, 37 ℃ digested 0.5 hour; Add the DMEM contain 5% calf serum and stop digestion, softly blow and beat mixing after 200 order cells sieve filter collecting cell; Under 1000 rev/mins of conditions centrifugal 5 minutes; Platform is expected blue dyeing counting viable cell, and adjusting cell density is 1 * 10 ⁶/ ml is seeded to the 25cm2 culturing bottle, adds the DMEM substratum that contains 10ng/ml bFGF and 10%FBS; Put 37 ℃, the CO of saturated humidity, volume fraction 5% ₂Cultivate in the incubator, inverted phase contrast microscope is observed growing state and the morphological specificity of former generation and passage cell lower every day, changes every other day liquid 1 time, and takes the photograph the sheet record; After cell degree of converging reaches 80%, digested 2-3 minute in 37 ℃ with 0.25% trypsinase-0.02%EDTA (ethylenediamine tetraacetic acid (EDTA)) solution, add substratum and stop trypsin acting, 1000 rev/mins were descended centrifugal 5 minutes, abandon supernatant, after cell precipitation is used the substratum Eddy diffusion, with 1 * 10 ⁷The cell density of/ml goes down to posterity; In the process that goes down to posterity, the next day change liquid 1 time, treat to repeat above step at the bottom of cell covers with 70% bottle;

The former culture step of described hAMSCs comprises: the amnion cell suspension is moved into centrifuge tube, trim, 2000 rev/mins carry out 15 minutes centrifugal, abandon supernatant, collecting cell; The cell of collecting is blown and beaten into even suspension with 0.01M PBS 30mL, 1500 rev/mins carry out 15 minutes centrifugal, wash collecting cell 2 times; Draw FBS 3mL before the inoculating cell and add T75cm ²The type Tissue Culture Flask is put 37 ℃, 5%CO ₂, the saturated humidity incubator was hatched 30 minutes, and culturing bottle is coated with; Abandon coating buffer, with containing 20%FBS, the separating obtained cell of the resuspended digestion of DMEM/F12 complete culture solution of 4ng/ml Urogastron is blown and beaten evenly counting cells; Adjusting cell concn is 1.0 * 10 ⁶Cell/ml is inoculated in the T75cm after being coated with ²In the type Tissue Culture Flask, place 37 ℃, 5%CO ², to cultivate in the cell culture incubator of saturated humidity, phase microscope is dynamically observed; Cultivate after 4-5 days full dose and change liquid, discard not attached cell, half amount was changed liquid once in later every 3-4 days; When the observation of cell adherent growth was converged to 80-90%, with 0.25% trypsinase-0.01%EDTA digestion, the gained cell was primary cell.

The going down to posterity to cultivate with amplification step of described hAMSCs comprises: when observing former generation hAMSCs cell attachment and growing to 80-90% and converge, inhale and abandon original nutrient solution in the culturing bottle, draw 10ml0.01M PBS damping fluid and add gently washing in the culturing bottle, discard washing lotion; Add 0.25% trypsinase-0.01%EDTA Digestive system 1.0ml, soak and overflow at a covering bottle end, observe under the inverted microscope; Add 1.0ml FBS and end trysinization; Add 10ml 0.01M PBS and repeatedly blow and beat flushing, at room temperature 900 rev/mins carry out 10 minutes centrifugal; Abandoning supernatant adds the 10mlDMEM/F12 re-suspended cell, in 1: 2-1: the cultivation of going down to posterity of 3 ratios; Put 37 ℃, 5%CO ₂, cultivate in the cell culture incubator of saturated humidity, count 1st generation (P1) hAMSCs, when treating that Growth of Cells converges to 80-90%, repeat aforesaid operations and carry out P2, P3, P4 ... increase for cell cultures.

The detecting step of described hAMSCs Immunophenotyping comprises: get cultivate the 2nd or the 3rd generation cell, at first use 0.25% tryptic digestion after, then adjust cell concn to 1 * 10 with the PBS that contains 1% bSA ⁷/ ml; Add respectively following mouse-anti human monoclonal antibodies: CD34-FITC, CD45-PE, CD19-FITC, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC, HLA-DR-FITC, putting 4 ℃ hatches half an hour, PBS washes 1 time, carries out flow cytometer and detects analysis.

The step that described hAMSCs is induced to differentiate into neuron cell comprises: get the 2-3 subtituted culturing cell, be inoculated in respectively 6 orifice plates, when stand density reaches 60%, change inducing culture into; 37 ℃, 5%CO ₂, saturated humidity leaves standstill cultivation.

Described immunofluorescent staining step comprises: culturing cell is inducing differentiation to adopt immunofluorescence dyeing after 3 days.

The present invention uses all-trans-retinoic acid (ATRA), Prostatropin (bFGF), Urogastron (EGF) and induces human amnion mesenchymal stem cell (hAMSCs) to be divided into neuron cell, the representative configuration that has not only had neurocyte, and expression neurone sign antigen neuronspecific enolase (neuron specific enolase, NSE), star spongiocyte sign antigen glial fibrillary acidic protein (glial fibrilament acidicprotein, GFAP); Point out BMSCs still to keep and stride the ability that differentiation of germinal layers is non-mesenchymal cell, can stride differentiation of germinal layers is neuron cell, is expected to become the seed cell of neurocyte replacement therapy.

Description of drawings

By the detailed description below in conjunction with accompanying drawing, aforesaid purpose, the feature and advantage with other of the present invention will become apparent.Wherein:

Figure 1 shows that human amnion mesenchymal stem cell of the present invention is divided into the flow chart of steps of the induction method of neuron cell.

Embodiment

Human amnion mesenchymal stem cell of the present invention as shown in Figure 1 is divided into the flow chart of steps of neuron cell induction method, and it is to use all-trans-retinoic acid, Prostatropin, Urogastron to induce human amnion mesenchymal stem cell to be divided into the described method of neuron cell and specifically may further comprise the steps:

The separation of S1, human amnion mesenchymal stem cell (hAMSCs):

Get the fresh human placenta of throwing aside postpartum under the aseptic condition, adopt mechanical process that amnion is peeled off from placenta tissue, for several times to remove residual bloodstain, the amnion after the rinsing is cut into approximately 1mm with eye scissors with the flushing of D-Hanks liquid ³Fragment adds 37 ℃ of digestion of 2.5g/L trypsinase 10 minutes; Add the DMEM that contains 5% calf serum and stop digestion, and filter with 200 order cells sieve after softly blowing and beating mixing; Add 1.0g/L II Collagenase Type Digestive system in the amnion tissue after the filtration, 37 ℃ digested 0.5 hour; Add the DMEM contain 5% calf serum and stop digestion, softly blow and beat mixing after 200 order cells sieve filter collecting cell; Under 1000 rev/mins of conditions centrifugal 5 minutes; Platform is expected blue dyeing counting viable cell, and adjusting cell density is 1 * 10 ⁶/ ml is seeded to 25cm ²Culturing bottle adds the DMEM substratum that contains 10ng/ml bFGF and 10%FBS; Put 37 ℃, the CO of saturated humidity, volume fraction 5% ₂Cultivate in the incubator, inverted phase contrast microscope is observed growing state and the morphological specificity of former generation and passage cell lower every day, changes every other day liquid 1 time, and takes the photograph the sheet record; After cell degree of converging reaches 80%, digested 2-3 minute in 37 ℃ with 0.25% trypsinase-0.02%EDTA (ethylenediamine tetraacetic acid (EDTA)) solution, add substratum and stop trypsin acting, 1000 rev/mins were descended centrifugal 5 minutes, abandon supernatant, after cell precipitation is used the substratum Eddy diffusion, with 1 * 10 ⁷The cell density of/ml goes down to posterity; In the process that goes down to posterity, the next day change liquid 1 time, treat to repeat above step at the bottom of cell covers with 70% bottle.

The former culture of S2, hAMSCs:

The amnion cell suspension is moved into centrifuge tube, trim, 2000 rev/mins carry out 15 minutes centrifugal, abandon supernatant, collecting cell; The cell of collecting is blown and beaten into even suspension with 0.01M PBS 30mL, 1500 rev/mins carry out 15 minutes centrifugal, wash collecting cell 2 times; Draw FBS 3mL before the inoculating cell and add T75cm ²The type Tissue Culture Flask is put 37 ℃, 5%CO ₂, the saturated humidity incubator was hatched 30 minutes, and culturing bottle is coated with; Abandon coating buffer, with containing 20%FBS, the separating obtained cell of the resuspended digestion of DMEM/F12 complete culture solution of 4ng/ml Urogastron (EGF) is blown and beaten evenly counting cells; Adjusting cell concn is 1.0 * 10 ⁶Cell/ml is inoculated in the T75cm after being coated with ²In the type Tissue Culture Flask, place 37 ℃, 5%CO ², to cultivate in the cell culture incubator of saturated humidity, phase microscope is dynamically observed; Cultivate after 4-5 days full dose and change liquid, discard not attached cell, half amount was changed liquid once in later every 3-4 days; When the observation of cell adherent growth was converged to 80-90%, with 0.25% trypsinase-0.01%EDTA digestion, the gained cell was primary cell.

Going down to posterity of S3, hAMSCs cultivated and amplification:

When observing former generation hAMSCs cell attachment and growing to 80-90% and converge, inhale and abandon original nutrient solution in the culturing bottle, draw 10ml 0.01M PBS damping fluid and add gently washing in the culturing bottle, discard washing lotion; Add 0.25% trypsinase-0.01%EDTA Digestive system 1.0ml, soak and overflow to cover a bottle end, observe under the inverted microscope, see that the intercellular substance increases, the kytoplasm retraction is seen the rounded levitating of cell after shaking piping and druming; Add 1.0ml FBS and end trysinization; Add 10ml 0.01M PBS and repeatedly blow and beat flushing, at room temperature 900 rev/mins carry out 10 minutes centrifugal; Abandoning supernatant adds 10ml DMEM/F12 re-suspended cell, in 1: 2-1: the cultivation of going down to posterity of 3 ratios; Culture system is 20%FBS, the DMEM/F12 complete culture solution 15ml/ bottle of 4ng/ml EGF.Put 37 ℃, 5%CO ₂, cultivate in the cell culture incubator of saturated humidity, count 1st generation (passage l, P1) hAMSCs, when treating that Growth of Cells converges to 80-90%, repeat aforesaid operations and carry out P2, P3, P4 ... increase for cell cultures.

The detection of S4, hAMSCs Immunophenotyping:

Get cultivate the 2nd or the 3rd generation cell, at first use 0.25% tryptic digestion after, then adjust cell concn to 1 * 10 with the PBS that contains 1% bSA ⁷/ ml; Add respectively following mouse-anti human monoclonal antibodies: CD34-FITC, CD45-PE, CD19-FITC, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC, HLA-DR-FITC, putting 4 ℃ hatches half an hour, PBS washes 1 time, carries out flow cytometer and detects analysis; The flow cytometer detection display, BMSCs expresses CD29, CD44 and CD106.

S5, hAMSCs are induced to differentiate into neuron cell:

Get the 2-3 subtituted culturing cell, be inoculated in respectively 6 orifice plates, when stand density reaches 60%, change inducing culture (DMEM+10%FBS+1 μ mol/L ATRA+20ng/ml bFGF+20ng/ml EGF) into; 37 ℃, 5%CO ₂, saturated humidity leaves standstill cultivation; Add induced liquid after 2 hours cellular form be considerable change, the tenuigenin of BMSCs shrinks to nuclear under the light microscopic, is typical perikaryon form; Most cells can form the neuron cell form after 3～5 hours, and cell space is rounded, and projection is longer, branch occurs in lug tips, and the projection of part flanking cell connects into net, but cell number is without obvious increase; Most cells changes into bipolar or multipolar neuron cell sample form after 3 days, stretches out cynapse (similar aixs cylinder or dendron), pulls into netted between the part cell.

S6, immunofluorescent staining:

Culturing cell is inducing differentiation to adopt immunofluorescence dyeing after 3 days.Attached cell removes substratum, wash 3 times with PBS, fix 30 minutes through 4% Paraformaldehyde 96, PBS washing 3 times, 0.3%TritonX-100 processed 20 minutes, PBS rinsing 3 times, 10% sheep blood serum room temperature sealing 20 minutes, sucking-off serum adds respectively the anti-human NSE of rabbit (1: 500), GFAP (1: 1000), hatches 2 hours for 37 ℃, then PBS washes 3 times, add sheep anti mouse-FITC (1: 500) and incubated 30 minutes, dye visible 50-70%NSE, 25-50%GFAP are positive, and the Nestin positive cell drops to 1.6% when 48h.

The present invention uses all-trans-retinoic acid (ATRA), Prostatropin (bFGF), Urogastron (EGF) is induced human amnion mesenchymal stem cell (human amnionmembrane mesenchymal stem cells, hAMSCs) be divided into neuron cell, the representative configuration that has not only had neurocyte, and expression neurocyte marker protein, prompting BMSCs has still kept the ability that differentiation of germinal layers is non-mesenchymal cell of striding, can stride differentiation of germinal layers is neuron cell, is expected to become the seed cell of neurocyte replacement therapy.

The present invention is not limited to described embodiment, and those skilled in the art still can do some corrections or change, therefore the scope of the present invention is as the criterion with claims restricted portion not breaking away from spirit of the present invention namely openly in the scope.

Claims (2)

1. a human amnion mesenchymal stem cell is divided into the neuron cell induction method, it is characterized in that, it uses All-trans retinoic acid plus bFGF, the EGF inducing umbilical cord mesenchymal stem is divided into neural stem cell, said method comprising the steps of: hAMSCs separates, the former culture of hAMSCs, going down to posterity of hAMSCs cultivated and amplification, the detection of hAMSCs Immunophenotyping, and hAMSCs is induced to differentiate into neuron cell and immunofluorescent staining; Wherein,

The step that described hAMSCs separates comprises: get the fresh human placenta of throwing aside postpartum under the aseptic condition, adopt mechanical process that amnion is peeled off from placenta tissue, with the flushing of D-Hanks liquid, the amnion after the rinsing is cut into approximately 1mm with eye scissors ³Fragment adds 37 ℃ of digestion of 2.5g/L trypsinase 10 minutes; Add the DMEM contain 5% calf serum and stop digestion, softly blow and beat behind the mixing with the filtration of 200 order cells sieve; Add 1.0g/L II Collagenase Type Digestive system in the amnion tissue after the filtration, 37 ℃ digested 0.5 hour; Add the DMEM contain 5% calf serum and stop digestion, softly blow and beat mixing after 200 order cells sieve filter collecting cell; Under 1000 rev/mins of conditions centrifugal 5 minutes; Platform is expected blue dyeing counting viable cell, and adjusting cell density is 1 * 10 ⁶/ ml is seeded to 25cm ²Culturing bottle adds the DMEM substratum that contains 10ng/ml bFGF and 10%FBS; Put 37 ℃, the CO of saturated humidity, volume fraction 5% ₂Cultivate in the incubator, inverted phase contrast microscope is observed growing state and the morphological specificity of former generation and passage cell lower every day, changes every other day liquid 1 time, and takes the photograph the sheet record; After cell degree of converging reaches 80%, digested 2-3 minute in 37 ℃ with 0.25% trypsinase-0.02%EDTA (ethylenediamine tetraacetic acid (EDTA)) solution, add substratum and stop trypsin acting, 1000 rev/mins were descended centrifugal 5 minutes, abandon supernatant, after cell precipitation is used the substratum Eddy diffusion, with 1 * 10 ⁷The cell density of/ml goes down to posterity; In the process that goes down to posterity, the next day change liquid 1 time, treat to repeat above step at the bottom of cell covers with 70% bottle;

The former culture step of described hAMSCs comprises: the amnion cell suspension is moved into centrifuge tube, trim, 2000 rev/mins carry out 15 minutes centrifugal, abandon supernatant, collecting cell; The cell of collecting is blown and beaten into even suspension with 0.01M PBS 30mL, 1500 rev/mins carry out 15 minutes centrifugal, wash collecting cell 2 times; Draw FBS 3mL before the inoculating cell and add T75cm ²The type Tissue Culture Flask is put 37 ℃, 5%CO ₂, the saturated humidity incubator was hatched 30 minutes, and culturing bottle is coated with; Abandon coating buffer, with containing 20%FBS, the separating obtained cell of the resuspended digestion of DMEM/F12 complete culture solution of 4ng/ml Urogastron is blown and beaten evenly counting cells; Adjusting cell concn is 1.0 * 10 ⁶Cell/ml is inoculated in the T75cm after being coated with ²In the type Tissue Culture Flask, place 37 ℃, 5%CO ², to cultivate in the cell culture incubator of saturated humidity, phase microscope is dynamically observed; Cultivate after 4-5 days full dose and change liquid, discard not attached cell, half amount was changed liquid once in later every 3-4 days; When the observation of cell adherent growth was converged to 80-90%, with 0.25% trypsinase-0.01%EDTA digestion, the gained cell was primary cell;

The going down to posterity to cultivate with amplification step of described hAMSCs comprises: when observing former generation hAMSCs cell attachment and growing to 80-90% and converge, inhale and abandon original nutrient solution in the culturing bottle, draw 10ml0.01M PBS damping fluid and add gently washing in the culturing bottle, discard washing lotion; Add 0.25% trypsinase-0.01%EDTA Digestive system 1.0ml, soak and overflow at a covering bottle end, observe under the inverted microscope; Add 1.0ml FBS and end trysinization; Add 10ml 0.01M PBS and repeatedly blow and beat flushing, at room temperature 900 rev/mins carry out 10 minutes centrifugal; Abandoning supernatant adds 10ml DMEM/F12 re-suspended cell, in 1: 2-1: the cultivation of going down to posterity of 3 ratios; Put 37 ℃, 5%CO ₂, cultivate in the cell culture incubator of saturated humidity, count 1st generation (P1) hAMSCs, when treating that Growth of Cells converges to 80-90%, repeat aforesaid operations and carry out P2, P3, P4 ... increase for cell cultures;

The detecting step of described hAMSCs Immunophenotyping comprises: get cultivate the 2nd or the 3rd generation cell, at first use 0.25% tryptic digestion after, then adjust cell concn to 1 * 10 with the PBS that contains 1% bSA ⁷/ ml; Add respectively following mouse-anti human monoclonal antibodies: CD34-FITC, CD45-PE, CD19-FITC, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC, HLA-DR-FITC, putting 4 ℃ hatches half an hour, PBS washes 1 time, carries out flow cytometer and detects analysis;

The step that described hAMSCs is induced to differentiate into neuron cell comprises: get the 2-3 subtituted culturing cell, be inoculated in respectively 6 orifice plates, when stand density reaches 60%, change inducing culture into; 37 ℃, 5%CO ₂, saturated humidity leaves standstill cultivation; And

Described immunofluorescent staining step comprises: culturing cell is inducing differentiation to adopt immunofluorescence dyeing after 3 days.

2. human amnion mesenchymal stem cell as claimed in claim 1 is divided into the neuron cell induction method, it is characterized in that, the inducing culture that described hAMSCs is induced to differentiate in the step of neuron cell is DMEM+10%FBS+1 μ mol/LATRA+20ng/ml bFGF+20ng/ml EGF.

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