CN115029315A - Application of MAPK signal pathway inhibitor VX-702 to improvement of hematopoietic differentiation potential of hESCs - Google Patents
Application of MAPK signal pathway inhibitor VX-702 to improvement of hematopoietic differentiation potential of hESCs Download PDFInfo
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Abstract
The invention relates to application of an MAPK signal pathway inhibitor VX-702 in improvement of hematopoietic differentiation potential of hESCs, belonging to the field of biotechnology. A method for efficiently and conveniently promoting the production of hematopoietic progenitor cells comprises the following steps: during the culture and maintenance stage of the human pluripotent stem cells, MAPK signal pathway inhibitor VX-702 is added into a culture dish, the cells after continuous treatment are subjected to in-vitro hematopoietic differentiation during the continuous treatment in the maintenance and culture stage, compared with untreated cells, the yield of blood progenitor cells is remarkably improved, and finally the yield of terminally differentiated blood cells is correspondingly improved. The MAPK signal pathway inhibitor VX-702 can promote the hematopoietic differentiation efficiency of hESCs in an in vitro hematopoietic differentiation system.
Description
Technical Field
The invention discloses an application of MAPK signal pathway inhibitor VX-702 in improvement of hematopoietic differentiation potential of hESCs, belongs to the field of biotechnology, and particularly relates to an application method for promoting the hematopoietic differentiation potential of human embryonic stem cells (hESCs) and improving the generation efficiency of hematopoietic progenitor cells by using a small-molecule inhibitor in the research of the culture process of pluripotent stem cells.
Background
The existing effective method for improving the hematopoietic differentiation efficiency of the in vitro hematopoietic differentiation system mainly changes the expression of certain hematopoietic differentiation key genes or changes the hematopoietic microenvironment in vitro. Inducible overexpression of gene modification can alter the cell transcriptional profile to promote hematopoietic progenitor cell production, which has the advantages of: the gene expression can be regulated at a specific time and a specific space, but the method also has some significant disadvantages, such as time and power consumption for constructing the transgenic cell line, potential canceration risk caused by random insertion of genes into the cell genome, and different regulation of gene expression quantity of the transgenic cell line established by different batches and clones. These are all uncontrollable factors, which affect the stability of the system.
At present, a set of methods for directional differentiation of human embryonic stem cells into hematopoietic stem/progenitor cells and mature blood cells in a laboratory is established, which comprises the following steps: the human pluripotent stem cell line is maintained in undifferentiated culture and co-cultured with AGM-S3 to generate hematopoietic stem/progenitor cells, which are then directionally induced to differentiate into mature blood cells including eosinophils, mast cells, erythrocytes, macrophages, etc. by liquid suspension culture. To date, this approach has been widely used to study the cytological/molecular mechanisms involved in the transition of pluripotent stem cells to hematopoietic differentiation. The laboratory observes the effect and function of the gene or the signal channel possibly exerted in the process of differentiating the human pluripotent stem cells into hematopoiesis by screening and regulating the expression of the key gene and the state of the key signal channel in the process of culturing the human pluripotent stem cells.
The research of hematopoietic differentiation uses human pluripotent stem cells (hPSC) as the starting cell source, and the cell lines all have the characteristic of strong silent vector expression exogenous gene and are cell types which are difficult to construct transgenic cell lines; in the process of differentiating hPSC into hemopoietic cells, many cell types appear, and the induction over-expression of genes needs to be realized in different cell types and development stages, which puts very strict requirements on vector systems.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides an application method of a MAPK signal pathway inhibitor VX-702 in improving the hematopoietic differentiation potential of hESCs. The method is a method which is simpler to operate, quicker and more economical compared with a transgenic method to achieve the aim of promoting the generation of hematopoietic progenitor cells, and is a novel method for improving the hematopoietic differentiation potential of hPSC by using a small molecule inhibitor.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
an application of MAPK signal pathway inhibitor VX-702 in improving the hematopoietic differentiation potential of hESCs.
The MAPK signal pathway inhibitor VX-702 can promote the hematopoietic differentiation efficiency of hESCs in an in vitro hematopoietic differentiation system.
A method for promoting the generation of hematopoietic progenitor cells efficiently and conveniently, which comprises the following steps:
during the maintenance culture stage of the human pluripotent stem cells, MAPK signal pathway inhibitor VX-702 is added into a culture dish, and the hESC cells after continuous treatment are subjected to in-vitro hematopoietic differentiation in the maintenance culture stage, so that compared with untreated cells, the yield of blood progenitor cells is remarkably improved, and finally, the yield of terminally differentiated blood cells is correspondingly improved.
Preferably, the progenitor cells are hematopoietic progenitor cells, myeloid progenitor cells, erythroid progenitor cells.
As a preferred embodiment herein, the MAPK signaling pathway inhibitor VX-702 is added to the culture dish at a final concentration of 1-7.5nM, more preferably 5 nM.
In a preferred embodiment of the present application, the duration of the treatment in the maintenance phase is 5 days.
Further, the MAPK signaling pathway inhibitor VX-702 has the formula:
as a preferred embodiment herein, the MAPK signaling pathway inhibitor VX-702 is formulated by the steps of: the VX-702 powder is dissolved in DMSO at a stock concentration of 5. mu.M, and then frozen at-80 ℃ for use.
Compared with the prior art, the positive effects of the invention are as follows:
the small molecule inhibitor is easy to obtain and simple to prepare. The small molecule inhibitor powder is purchased from a company and dissolved in DMSO to be prepared into the required concentration.
And (II) the operation is simple, the reagent is directly added into the culture medium, the operations of transfecting cells, establishing cell lines and the like are not needed, and the cell state is basically not influenced or canceration is generated.
And thirdly, the working concentration of the small molecule inhibitor is not high, the small molecule inhibitor has no obvious toxicity to cells, and the cost is lower.
And fourthly, the hematopoietic differentiation potential of the hESC can be comprehensively improved, and the hESC has a promoting effect on various hematopoietic lineages, and is suitable for improving hematopoietic differentiation systems of different in-vitro hematopoietic differentiation systems in principle.
Description of the drawings:
FIG. 1 is a diagram showing the results of flow cytometry in the examples;
FIG. 2 is a graph showing the results of colony culture in the examples;
FIG. 3 is a graph showing the results of the cell sorting experiment in the examples;
Detailed Description
The present invention will be described in further detail with reference to specific embodiments for making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples.
The related materials such as the reagent kit and the like in the following examples are all commercial products; the MAPK signaling pathway inhibitor VX-702 has the formula:
a material
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Superclean bench LABCONCO A2
CO 2 Incubator Thermo HERACSL SD
Ordinary inverted microscope OLYMPUS CKX31
Flow cytometer BDFACSCAntoII/Calibur
Thermo LABOFUGE 400 of table type normal temperature high speed centrifuge
Biological irradiator RADSURE RS-2000
Adherent 12-orifice plate CORNING
(2) Primary reagent
BSA Sigma A9418 USA
IMDM Medium U.S. GIBCO 12440053
DMEM Medium Sigma D5796 USA
F12 culture fluid U.S. GIBCO 21700-075
KSR serum U.S. Gibco N10828-028
mTeSR1 Medium U.S. STEM CELL 05850
Y-27632(ROCK inhibitor) Germany MERCK 688000
Modified alpha-MEM US Hyclone SH30265.01B
Nonessential amino acids (NEAA) U.S. Gibco07796
Sigma beta-mercaptoethanol USA
0.25% Trypsin/EDTA (WT/V) U.S. Gibco 25200056
Fetal Bovine Serum (FBS) Hyclone 0078 of USA
bFGF Japanese WAKO 060-
Vascular endothelial cell growth factor (VEGF) Japanese WAKO 229-01313
L-Glutamine U.S. Gibco 25030081
M-penicillin/streptomycin Gibco 15070063
May-Giemsa dye liquor German MERCK
Trypan blue (TrypanBluestain) Gibco Inc. in USA
Ascorbic acid (ascorbic acid) Sigma USA
Transferrin (Transferrin) Sigma USA
Matrigel American BD Co
(3) Flow antibody and 7-AAD dye were purchased from BD corporation, usa:
2. preparation of the principal solution
(1) Preparation of humanized ESC culture medium
mTeSR TM 1 Basal Medium 400mL
mTeSR TM 1 5◇ Supplement 100mL
500ml in total, mixed evenly and placed in a refrigerator at 4 ℃ for standby.
(2) Preparation of AGM culture solution
α-MEM 500mL
FBS (Desplementization) 25mL
525ml, mixing, filtering with 0.22 μm filter, and storing in 4 deg.C refrigerator. (3) hESC undifferentiated cell maintenance liquid preparation
A total of 630ml, mixed well, filtered through a 0.22 μm filter and placed in a refrigerator at 4 ℃ for later use.
(4) Preparation of hematopoietic differentiation inducing liquid
590ml in total, mixed evenly, filtered by a 0.22 mu m filter and placed in a refrigerator at 4 ℃ for standby.
3, cell line
(1) H1 hESCs was supplied by the national focus laboratory of laboratory hematology of the Chinese academy of medical sciences;
(2) AGM-S3 was maintained in the laboratory.
4 MAPK Signal pathway inhibitor VX-702, purchased from Selleck reagent Inc
1. Formulation of MAPK Signal pathway inhibitors (VX-702)
VX-702 powder was dissolved in DMSO at a stock concentration of 5. mu.M in a clean bench and stored in aliquots frozen at-80 ℃.
2. Cell culture and passage of human pluripotent stem cell line
The human embryonic Stem cell line used in this study was H1 hESC in mTeSR1 medium (Stem cell Co.) at 37 deg.C with 5% CO 2 (V/V) in an incubator. After the cells had grown to 80% confluence, the cells were washed once with D-PBS, digested with ReleSR (Stem cell, cat:05872) for 5 minutes at 37 ℃, and the cell pellet was blown into appropriate small pieces with mTeSR1 medium and subcultured to Matrigel (Invitrogen, cat: A1413301) bottomed dishes at an area ratio of 1: 3.
VX-702 treatment of human pluripotent Stem cells
H1 hESCs were seeded into Matrigel-primed 24-well plates at a culture area ratio of 1:3 and cultured for 1-2 days, when hESC clones reached the appropriate size, either the corresponding volume of VX-702 was added to a working concentration of 5nM, or an equal volume of DMSO was added as a control, both for 5 days of continuous treatment of H1 hESCs, during which time the passaging was performed normally if the cells were overgrown.
4. The co-culture system examined the effect of VX-702 on the hematopoietic differentiation potential of human pluripotent stem cells.
(1) Preparation of AGM-S3 stromal cells
The AGM-S3 cell line frozen in liquid nitrogen was thawed in a gelatin-coated 100mm petri dish in the presence of MEM ALPHA MOD (SH30265.01, Hyclone) containing 5% fetal bovine serum (V/V), 37 deg.C, 5% CO 2 (V/V) culture until cells are completely confluent, then resuspension is digested with 0.05% pancreatic enzymes (WT/V), digestion is stopped with ice D-PBS and medium, passaged into gelatin-coated 12-well plates, gently shaken, and 5% CO at 37 deg.C 2 (V/V) culture for 1-2 days. When the degree of cell fusion is not less than 90% (about 2X 10) 5 Cells/well), 13Gray X-ray irradiation treatment for use.
(2) Co-culture of hESCs treated with VX-702 with AGM-S3 gives rise to hematopoietic progenitor cells
The hESCs undifferentiated colonies were microscopically divided into 0.5-1X 10 colonies with a 200. mu.L tip 3 Small colony pieces of cells, with 35 colonies/12 well plate per well (about 2X 10) 5 hESCs/well) on irradiated AGM-S3 cells, 1.5ml of hESCs undifferentiated cell maintenance fluid, gently shakingAnd culturing for 3 days, and then changing into hematopoietic differentiation inducing solution. At this point, marked as D0, and then cultured for 14 days with fluid changes each day, large numbers of hematopoietic stem/progenitor cells and erythroid/myeloid progenitor cells will be produced during the period from D8 to D14, and the cells will be collected for use.
(3) Flow cytometry analysis
Washing co-cultured cells of D8 and D14 with D-PBS for 2 times, treating with 0.25% Trypsin/EDTA (WT/V) at 37 deg.C for 5-7min, stopping pancreatin action with ice D-PBS and serum-containing culture solution, repeatedly blowing with 1ml gun head until no large cell mass agglutinates, collecting into centrifuge tube, centrifuging at 1300rpm for 5min at room temperature, discarding supernatant, and resuspending in SM buffer (D-PBS containing 2% FBS (V/V)), 70 μm
Filtering with a filter membrane. Approximately every 0.5ml of common rabbit serum was added in 5-10. mu.l and mixed, placed on ice for 20 minutes for blocking antibody antigen non-specific binding sites, 3. mu.l each of the corresponding flow antibodies was added, mixed by shaking, incubated on ice for 20-30 minutes in the absence of light, washed 1 time with 1ml D-PBS, centrifuged at 400. mu.g at room temperature for 5min, the supernatant was discarded, 200. mu.l of SM buffer solution was resuspended in a flow-up tube, examined using a BD CantoII flow cytometer, and the experimental data were analyzed using Flowjo10 software.
5. Colony culture detection of effect of VX-702 on promotion of hESC hematopoietic differentiation potential
Human pluripotent stem cells were maintained in culture for a period of 5 days with a final concentration of 5nM of the MAPK signaling pathway inhibitor VX-702 or an equal volume of DMSO for 5 days, and then co-cultured with AGM-S3, respectively. 3 ten thousand cells were taken out at the day 14 of co-culture and subjected to hematopoietic colony formation assay again, and the number of CFU-E (erythrocyte colony) was counted on day 7 after colony culture, and the number of CFU-Mix (mixed type colony), CFU-GM (neutrophil type colony), and BFU-E (explosive erythrocyte colony) were counted on days 12 to 14.
6. Sorting KDR + cells for suspension hematopoietic culture and detecting effect of VX-702 on promotion of hESC hematopoietic differentiation potential
Human pluripotent stem cells were maintained in culture for a period of 5 days with a final concentration of 5nM of the MAPK signaling pathway inhibitor VX-702 or an equal volume of DMSO for 5 days, and then co-cultured with AGM-S3, respectively. KDR + cell populations were individually sorted on day 4 of co-culture, and then co-cultured with AGM-S3 for additional flow cytometry detection on day 12.
The following example operations were carried out using the materials and process preparations of the specific embodiments:
example 1:
the application of VX-702, an MAPK signaling pathway inhibitor, in improving the hematopoietic differentiation potential of hESCs.
Example 2:
the MAPK signaling pathway inhibitor VX-702 can promote the efficiency of hematopoietic differentiation in an in vitro hematopoietic differentiation system.
Example 3:
a method for efficiently and conveniently promoting the production of hematopoietic progenitor cells comprises the following steps:
in the maintenance culture stage of the human pluripotent stem cells, MAPK signal pathway inhibitor VX-702 is respectively added into a culture dish, and the cells after continuous treatment and AGM-S3 cells are cultured together in the continuous treatment in the maintenance culture stage, which comprises the following specific operations:
human pluripotent stem cells maintain the culture phase: h1 hESCs were treated continuously for 5 days with MAPK signaling pathway inhibitor VX-702 or an equal volume of DMSO at final concentrations of 5, 7.5 or 10nM, respectively, and then co-cultured with the mouse stromal cell line AGM-S3, respectively, to differentiate into hematopoiesis. The specific result is shown in figure 1, and figure 1 is a flow cytometry detection result graph; after the cocultured cells are incubated and combined with the anti-CD 34/CD43 antibody after 8 days of hematopoietic differentiation induction in the step (A), the detection result of a flow analyzer shows that the ratio of CD34-CD43+ and CD34+ CD43+ cell populations in the cocultured cells is obviously increased when 5nM VX-702 is used for treating the hESC compared with a control group. (B) After the co-cultured cells are incubated and combined with anti-CD 34/CD43/CD45 antibody after 14 days of hematopoietic differentiation induction, the flow analysis and detection result shows that the proportion of cell populations such as CD34+ CD43+ (hematopoietic progenitor cell population), CD34+ CD45+ (myeloid progenitor cell population) and GPA + CD71+ (erythroid progenitor cell population) in the co-cultured cells is obviously improved when 5nM VX-702 is used for treating hESC compared with the control group. The above results clearly show that 5nM VX-702 has a strong promoting effect on the production of hematopoietic progenitor cells, myeloid progenitor cells and erythroid progenitor cells.
Example 4:
human pluripotent stem cells were maintained in culture for 5 days with a final concentration of 5nM MAPK signaling pathway inhibitor VX-702 or an equal volume of DMSO of H1 hESCs, and then co-cultured with mouse stromal cells AGM-S3, respectively. 3 ten thousand cells were taken out at the day of co-culture for colony culture again, and the numbers of CFU-E (erythrocyte colony) were counted on the 7 th day after colony culture, and CFU-Mix (mixed type colony), CFU-GM (neutrophil type colony), and BFU-E (explosive erythrocyte colony) were counted on the 12 th to 14 th days. The specific results are shown in FIG. 2, and FIG. 2 is a graph of colony culture results; wherein (A) is the number of colonies formed after treatment with VX-702 or DMSO. (B) Morphology of different colonies under microscope. The results show that 5nM VX-702 treatment promoted the production of CFU-E, BFU-E, CFU-GM colonies.
Example 5:
human pluripotent stem cells were maintained in culture for 5 days with a final concentration of 5nM of MAPK signaling pathway inhibitor VX-702 or an equal volume of DMSO, and then co-cultured with mouse stromal cells AGM-S3, respectively. KDR + cell populations were individually sorted on day 4 of co-culture, then co-cultured with AGM-S3 cells, and flow cytometry detection was performed on day 12. The specific results are shown in FIG. 3, and FIG. 3 is a graph of the results of the cell sorting experiment; flow assays in which (A) and (B) VX-702 or equal volumes of DMSO were treated prior to sorting KDR + cell populations and re-co-culturing with AGM-S3. (C) Histogram after count correction of flow cell data.
The results indicate that 5nM VX-702 treated hESCs significantly promoted the production of CD34+ CD 43-and CD34-CD43+ cell populations during the pluripotent stem cell culture phase.
The above examples demonstrate that the yields of hematopoietic progenitor cells, myeloid progenitor cells and erythroid progenitor cells are significantly improved at different periods of co-culture compared to the control; the inhibitor can improve the hematopoietic differentiation potential of hESCs and promote the hematopoietic differentiation efficiency in an in vitro hematopoietic differentiation system.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. An application of MAPK signal pathway inhibitor VX-702 in improving the hematopoietic differentiation potential of hESCs.
2. The use of claim 1, wherein: the MAPK signal pathway inhibitor VX-702 can promote the hematopoietic differentiation efficiency of hESCs in an in vitro hematopoietic differentiation system.
3. A method for efficiently and conveniently promoting the production of hematopoietic progenitor cells is characterized by comprising the following steps:
adding MAPK signal pathway inhibitor VX-702 into a culture dish in a maintenance culture stage of the human pluripotent stem cells, continuously treating in the maintenance culture stage, and performing in-vitro hematopoietic differentiation on the continuously treated cells; the production of blood progenitor cells is significantly increased compared to untreated cells and ultimately results in a corresponding increase in the production of terminally differentiated blood cells.
4. The method of claim 3, wherein: the blood progenitor cells are hematopoietic progenitor cells, myeloid progenitor cells and erythroid progenitor cells.
5. The method of claim 3, wherein: the final concentration of MAPK signaling pathway inhibitor VX-702 added to the dishes ranged from 1-7.5 nM.
6. The method of claim 3, wherein: a typical final concentration of the MAPK signaling pathway inhibitor VX-702 added to the dishes was 5 nM.
7. The method of claim 3, wherein: the duration of treatment during the maintenance culture phase was 5 days.
8. The method of claim 3, wherein: the MAPK signaling pathway inhibitor VX-702 has the chemical formula:
9. the method of claim 6, wherein: the MAPK signal pathway inhibitor VX-702 is prepared by the following steps: the VX-702 powder was dissolved in DMSO at a stock concentration of 5. mu.M, and was frozen at-80 ℃ for use.
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| US20130171110A1 (en) * | 2011-12-05 | 2013-07-04 | DeWitt Ross & Stevens S.C. | Compositions and methods for differentiating pluripotent stem cells into primitive blood cells and uses thereof |
| CN109415699A (en) * | 2016-06-23 | 2019-03-01 | 国立大学法人京都大学 | The preparation method of the bis- positive T cells of CD4CD8 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20130171110A1 (en) * | 2011-12-05 | 2013-07-04 | DeWitt Ross & Stevens S.C. | Compositions and methods for differentiating pluripotent stem cells into primitive blood cells and uses thereof |
| CN109415699A (en) * | 2016-06-23 | 2019-03-01 | 国立大学法人京都大学 | The preparation method of the bis- positive T cells of CD4CD8 |
Non-Patent Citations (1)
| Title |
|---|
| NAFEES RAHMAN等: "Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence", 《NATURE COMMUNICATIONS》, vol. 8, pages 1 - 12 * |
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