Method for inducing pluripotent stem cells to be differentiated into CD34 positive hematopoietic endothelial cells
By using a feeder-free cell system and rhodioloside, the differentiation process of pluripotent stem cells was simplified, solving the problems of complex culture conditions and low efficiency in existing technologies, and achieving efficient and low-cost preparation of CD34-positive hematopoietic endothelial cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING JIANQIANG WEIYE TECH CO LTD
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies for in vitro hematopoietic differentiation of human pluripotent stem cells involve complex culture conditions, long differentiation cycles, low differentiation efficiency and yield, poor operability, and high costs. Furthermore, the use of serum and mouse stromal cells makes them unsuitable for clinical application.
Using a feeder-free cell system and the small molecule compound rhodioloside, oxidative damage was reduced through a specific mechanism, and culture conditions were simplified. Pluripotent stem cells were induced to differentiate into CD34-positive hematopoietic endothelial cells using D/F12 medium and rhodioloside.
It achieves efficient, simple, and rapid induction of human pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells, with stable differentiation effect, simplified operation process, reduced cost, and improved differentiation efficiency and yield.
Smart Images
Figure CN121950677A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells. Background Technology
[0002] Pluripotent stem cells include embryonic stem cells derived from embryos and induced pluripotent stem cells derived from in vitro reprogramming. Pluripotent stem cells possess the ability to maintain self-renewal through long-term in vitro culture and have the potential for multi-lineage differentiation, including differentiation into almost all functional blood cells. Hematopoietic endothelial cells are progenitor cells with self-renewal capacity and the ability to differentiate into various blood cells. However, obtaining a sufficient number of hematopoietic endothelial cells has long been a challenge for researchers and medical professionals. Since Kaufman et al. first successfully differentiated human embryonic stem cells into CD34+ hematopoietic endothelial cells in 2001 [Kaufman D, et al. 2001], research on the in vitro differentiation of human pluripotent stem cells into hematopoietic stem cells and related studies has been conducted. Maxim A. Vodyanik et al. reported a method for obtaining CD34-positive hematopoietic endothelial cells by co-culturing human pluripotent stem cells with mouse-derived stromal cells (or trophoblast cells) (Maxim A. Vodyanik et al., 2005). However, this method used fetal bovine serum and mouse stromal cells, making it unsuitable for clinical application. From a current clinical application perspective, research on the in vitro hematopoietic differentiation of human pluripotent stem cells still faces significant challenges. For example, the in vitro culture conditions for inducing differentiation of human pluripotent stem cells into hematopoietic endothelial cells involve exogenous substances such as serum and feeder cells, which greatly limits the current clinical application of in vitro hematopoietic differentiation using human pluripotent stem cells. Furthermore, current methods involve complex culture conditions, relatively long differentiation cycles, low differentiation efficiency and yield, and poor operability. Based on these problems with existing differentiation techniques, there is an urgent need to find a method for inducing hematopoietic endothelial cells. Summary of the Invention
[0003] To address the problems existing in the prior art, this invention provides a method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells, thereby solving the problems of complex culture conditions, long differentiation cycle, low differentiation efficiency and yield, poor operability, complex operation and high cost in existing methods for preparing hematopoietic stem cells.
[0004] The technical solution of the present invention is as follows: This invention provides a method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells, comprising the following steps: a. Culture pluripotent stem cells until the pluripotent stem cell confluence reaches 50-80%; b. Digest pluripotent stem cells into single cells, and resuspend the pluripotent stem cells in a culture medium to obtain a cell suspension; c. Place the cell suspension into a cell culture plate and culture it in an adherent manner; d. After the culture in step c is completed, discard the culture medium described in step b, add hematopoietic differentiation culture medium to the cell culture plate, and induce culture to form CD34 positive hematopoietic endothelial cells; The hematopoietic differentiation culture medium consists of D / F12 medium and rhodioloside.
[0005] Furthermore, the pluripotent stem cells are human pluripotent stem cells.
[0006] Furthermore, the CD34-positive hematopoietic endothelial cells indicate that the hematopoietic endothelial cells have the CD34+ phenotype.
[0007] Furthermore, in step a, the culture conditions are as follows: the culture is carried out in a cell culture incubator at 37°C containing 5% CO2, and the culture system is a cell system without a feeder layer. Specifically, the operation is as follows: mTeSR medium is added to a cell culture plate covered with matrix gel, pluripotent stem cells are seeded into the mTeSR medium, and the mTeSR medium is changed daily.
[0008] Furthermore, step b specifically includes: after the pluripotent stem cells have reached a confluence of 50-80%, digesting the pluripotent stem cells into single cells using accutase; then, distributing the pluripotent stem cells at a concentration of 1×10⁻⁶ cells / cells. 4 The cells were resuspended in mTeSR medium at a concentration of 1 cell / mL to obtain a cell suspension.
[0009] Furthermore, the confluence of the pluripotent stem cells is 80%.
[0010] Furthermore, in step c, the cell culture plate is pre-coated with matrix gel, and the culture conditions are: cultured in a cell culture incubator at 37°C containing 5% CO2.
[0011] Furthermore, the concentration of rhodioloside is 10-100 μmol / L.
[0012] Preferably, the concentration of rhodioloside is 100 μmol / L.
[0013] Furthermore, in step d, the method of discarding the culture medium mentioned in step b is to aspirate the culture medium by vacuum suction, and the conditions for induction culture are: cultured for 6 days in a cell culture incubator at 37°C containing 5% CO2.
[0014] The present invention also provides CD34-positive hematopoietic endothelial cells prepared by the above method.
[0015] The beneficial effects of this invention are as follows: This invention provides a method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells. This method uses a small molecule compound, rhodioloside, to efficiently, simply, and rapidly induce human pluripotent stem cells to differentiate into stable hematopoietic endothelial cells with a CD34+ phenotype. Mechanistically, the addition of rhodioloside during pluripotent stem cell differentiation reduces oxidative damage through a specific mechanism, creating a more stable environment for hematopoietic differentiation and inhibiting oxidative reactions. This invention provides hematopoietic endothelial cells with a CD34+ phenotype differentiated from pluripotent stem cells. This method has the advantages of simplified culture conditions, short differentiation cycle, high differentiation efficiency, strong operability, extremely simple operation, high yield, and low cost, and its main component is only a single small molecule compound, rhodioloside. This method also possesses significant safety, practicality, and reproducibility. This invention provides a new option for preparing CD34-positive hematopoietic endothelial cells. Attached Figure Description
[0016] Figure 1 The flowchart for inducing pluripotent stem cell differentiation into hematopoietic endothelial cells in vitro includes: Single cell seeding; DMEM / F12 medium. Figure 2 The graph shows the results of flow cytometry detection of CD34 differentiation efficiency in Example 1; Figure 3 This is a graph showing the results of flow cytometry analysis of CD34 differentiation efficiency in Example 2; Figure 4 This is a graph showing the results of flow cytometry analysis of CD34 differentiation efficiency in Example 3; Figure 5 The flowchart shows the existing technology for inducing pluripotent stem cell differentiation into hematopoietic endothelial cells in vitro, where Single cellseeding and Stempro34 SFM are serum-free Stempro34 culture media. Figure 6 The figure shows the results of flow cytometry analysis of CD34 differentiation efficiency in Comparative Example 1. Figure 7 The figure shows the results of flow cytometry analysis of CD34 differentiation efficiency in Comparative Example 2. Detailed Implementation
[0017] The present invention will be further described in detail below through embodiments, but in no way is the invention limited.
[0018] The mTeSR medium used in the examples was a commercially available medium purchased from Stem Cell Technologies; the D / F12 medium was a commercially available medium purchased from Beijing Jianqiang Weiye Technology Co., Ltd.; and rhodioloside was purchased from MCE (medchemexpress), catalog number HY-N0109.
[0019] From a mechanistic perspective, in the pluripotent stem cell differentiation process of Example 1, the addition of rhodioloside can reduce "oxidative damage" in the pluripotent stem cell differentiation process through a specific mechanism, creating a more stable environment for hematopoietic differentiation and inhibiting oxidative reactions.
[0020] Example 1: Rhodiola rosea induces human pluripotent stem cells to differentiate into hematopoietic endothelial cells. 1. Culture of human pluripotent stem cells (1) The culture system is a feederless cell system. Specifically, Matrigel is diluted 1:100 in PBS and then spread into a cell culture plate. Then, mTeSR medium is added to the cell culture plate with Matrigel. Pluripotent stem cells are seeded into mTeSR medium for maintenance and expansion. The cell culture plate is a six-well plate, and each well of the six-well plate contains 2 mL of mTeSR medium. (2) The maintenance and expansion process includes: placing human pluripotent stem cells in a 37°C cell culture incubator containing 5% CO2 for culture, and observing and changing the medium regularly every day; after the human pluripotent stem cells reach 80% confluence, they can be passaged.
[0021] 2. Directed differentiation of human pluripotent stem cells into hematopoietic endothelial cells The flowchart of the directed differentiation of human pluripotent stem cells into hematopoietic endothelial cells is as follows: Figure 1 As shown, the specific steps are as follows: (1) D-1 (Preparation stage one day before the start): After the confluence of human pluripotent stem cells reaches 80%, human pluripotent stem cells are digested into single cells using acutase and counted; 2×10 4 Human pluripotent stem cells were resuspended in 2 mL of mTeSR medium to obtain a cell suspension. The cell suspension was then seeded into one well of a six-well plate pre-coated with matrix gel. Finally, the human pluripotent stem cells were placed in a 37°C cell culture incubator containing 5% CO2 for adherent culture.
[0022] (2) D0 (starting day): Prepare hematopoietic differentiation medium: D / F12 medium + 10 μmol / L rhodioloside, where D / F12 medium is also called DMEM / F12 medium; then remove the old medium by vacuum aspiration and add 2 mL of hematopoietic differentiation medium to each well of human pluripotent stem cells; then continue to place human pluripotent stem cells in a 37℃ cell culture incubator containing 5% CO2 for six days to form hematopoietic endothelial cells.
[0023] (3) D6 (sixth day after the start date): Flow cytometry was used to detect CD34 expression. CD34 is a marker of hematopoietic endothelial cells, also known as hematopoietic endothelial cells or CD34+ hematopoietic endothelial cells. The results of flow cytometry detection of CD34 differentiation efficiency are as follows: Figure 2 As shown in the figure, the cells in the box are CD34 positive, accounting for 58.51%.
[0024] Example 2: Rhodiola rosea induces human pluripotent stem cells to differentiate into hematopoietic endothelial cells. 1. Culture of human pluripotent stem cells (1) The culture system is a feederless cell system. Specifically, Matrigel is diluted 1:100 in PBS and then spread into a cell culture plate. Then, mTeSR medium is added to the cell culture plate with Matrigel. Pluripotent stem cells are seeded into mTeSR medium for maintenance and expansion. The cell culture plate is a six-well plate, and each well of the six-well plate contains 2 mL of mTeSR medium. (2) The maintenance and expansion process includes: placing human pluripotent stem cells in a 37°C cell culture incubator containing 5% CO2 for culture, and observing and changing the medium regularly every day; after the human pluripotent stem cells reach 80% confluence, they can be passaged.
[0025] 2. Directed differentiation of human pluripotent stem cells into hematopoietic endothelial cells (1) D-1 (Preparation stage one day before the start): After the confluence of human pluripotent stem cells reaches 80%, human pluripotent stem cells are digested into single cells using acutase and counted; 2×10 4 Human pluripotent stem cells were resuspended in 2 mL of mTeSR medium to obtain a cell suspension. The cell suspension was then seeded into one well of a six-well plate pre-coated with matrix gel. Finally, the human pluripotent stem cells were placed in a 37°C cell culture incubator containing 5% CO2 for adherent culture.
[0026] (2) D0 (starting day): Prepare hematopoietic differentiation medium: D / F12 medium + 100 μmol / L rhodioloside, where D / F12 medium is also called DMEM / F12 medium; then remove the old medium by vacuum aspiration and add 2 mL of hematopoietic differentiation medium to each well of human pluripotent stem cells; then continue to place human pluripotent stem cells in a 37℃ cell culture incubator containing 5% CO2 for six days to form hematopoietic endothelial cells.
[0027] (3) D6 (sixth day after the start date): Flow cytometry was used to detect CD34 expression. CD34 is a marker of hematopoietic endothelial cells, also known as hematopoietic endothelial cells or CD34+ hematopoietic endothelial cells. The results of flow cytometry detection of CD34 differentiation efficiency are as follows: Figure 3 As shown in the figure, the cells in the box are CD34 positive, accounting for 64.4%.
[0028] Example 3: Rhodiola rosea induces human pluripotent stem cells to differentiate into hematopoietic endothelial cells. 1. Culture of human pluripotent stem cells (1) The culture system is a feederless cell system. Specifically, Matrigel is diluted 1:100 in PBS and then spread into a cell culture plate. Then, mTeSR medium is added to the cell culture plate with Matrigel. Pluripotent stem cells are seeded into mTeSR medium for maintenance and expansion. The cell culture plate is a six-well plate. Each well of the six-well plate contains 2 mL of mTeSR medium and 2 mL of Matrigel. (2) The maintenance and expansion process includes: placing human pluripotent stem cells in a 37°C cell culture incubator containing 5% CO2 for culture, and observing and changing the medium regularly every day; after the human pluripotent stem cells reach 80% confluence, they can be passaged.
[0029] 2. Directed differentiation of human pluripotent stem cells into hematopoietic endothelial cells (1) D-1 (Preparation stage one day before the start): After the confluence of human pluripotent stem cells reaches 80%, human pluripotent stem cells are digested into single cells using acutase and counted; 2×10 4 Human pluripotent stem cells were resuspended in 2 mL of mTeSR medium to obtain a cell suspension. The cell suspension was then seeded into one well of a six-well plate pre-coated with matrix gel. Finally, the human pluripotent stem cells were placed in a 37°C cell culture incubator containing 5% CO2 for adherent culture.
[0030] (2) D0 (starting day): Prepare hematopoietic differentiation medium: D / F12 medium + 50 μmol / L rhodioloside, where D / F12 medium is also called DMEM / F12 medium; then remove the old medium by vacuum aspiration and add 2 mL of hematopoietic differentiation medium to each well of human pluripotent stem cells; then continue to place human pluripotent stem cells in a 37℃ cell culture incubator containing 5% CO2 for six days to form hematopoietic endothelial cells.
[0031] (3) D6 (sixth day after the start date): Flow cytometry was used to detect CD34 expression. CD34 is a marker of hematopoietic endothelial cells, also known as hematopoietic endothelial cells or CD34+ hematopoietic endothelial cells. The results of flow cytometry detection of CD34 differentiation efficiency are as follows: Figure 4 As shown in the figure, the cells in the box are CD34 positive, accounting for 55.5%.
[0032] Comparative Example 1: Induced pluripotent stem cell 3D embryoid bodies undergo hematopoietic endothelial differentiation In existing technologies, the technical flowchart for inducing human pluripotent stem cells to differentiate into hematopoietic endothelial cells is as follows: Figure 5 As shown. The specific operation steps are as follows: 1. Single-cell preparation and embryoid formation D-1 (Preparation stage one day before start): Single-cell seeding of induced pluripotent stem cells is completed using mTeSR1 medium. First, the induced pluripotent stem cells are dissociated into single-cell suspensions, and then the single cells are aggregated to form embryoid bodies through suspension culture.
[0033] 2. Switching between culture medium and culture system The resulting embryoids were transferred to Stempro34 SFM to enter the "hematopoietic endothelial induction stage".
[0034] 3. Induction of hematopoietic endothelial growth by adding a combination of cytokines to Stempro34 medium in stages: D0 (Start Date): Add BMP4 to Stempro34 SFM; D1 (Day 1 after start date): Add a combination of cytokines BMP4 and bFGF to Stempro34 SFM; D1.75 (18 hours after the start date): Add a combination of cytokines BMP4, bFGF, CHIR99021 and SB431542 to Stempro34 SFM; D3 (the third day after the start date): Maintain the cytokine combination of BMP4, bFGF, CHIR99021 and SB431542 in Stempro34 SFM; D4 (fourth day after the start date): Add a combination of cytokines VEGF, IL-6, IL-11 and bFGF to Stempro34 SFM; D6 (sixth day after the start date): Add a combination of cytokines including VEGF, EPO, IGF-1, SCF, IL-6, IL-11 and bFGF to Stempro34 SFM.
[0035] The Chinese meanings of BMP4, bFGF, VEGF, IL-6, IL-11, EPO, IGF-1, and SCF mentioned above are as follows: BMP4: Bone morphogenetic protein 4; bFGF: Basic fibroblast growth factor; VEGF: Vascular endothelial growth factor; IL-6: Interleukin-6; IL-11: Interleukin-11; EPO: Erythropoietin; IGF-1: Insulin-like growth factor 1; SCF: Stem cell factor.
[0036] CHIR99021 and SB431542 are codes for small molecule inhibitors.
[0037] 4. On D8 (the eighth day after the start date), the differentiation results will be detected. On day 8 of induction, cells were collected and CD34 expression levels were detected by flow cytometry to determine the proportion of CD34+ hematopoietic endothelial cells obtained after induction (i.e., differentiation efficiency). The results are as follows: Figure 6 As shown in the figure, the cells in the box are CD34 positive, accounting for 20.33%.
[0038] Comparative Example 2: Hematopoietic endothelial differentiation of induced pluripotent stem cells without the addition of rhodioloside 1. Culture of human pluripotent stem cells (1) The culture system is a feederless cell system. Specifically, Matrigel is diluted 1:100 in PBS and then spread into a cell culture plate. Then, mTeSR medium is added to the cell culture plate with Matrigel. Pluripotent stem cells are seeded into mTeSR medium for maintenance and expansion. The cell culture plate is a six-well plate. Each well of the six-well plate contains 2 mL of mTeSR medium and 2 mL of Matrigel. (2) The maintenance and expansion process includes: placing human pluripotent stem cells in a 37°C cell culture incubator containing 5% CO2 for culture, and observing and changing the medium regularly every day; after the human pluripotent stem cells reach 80% confluence, they can be passaged.
[0039] 2. Directed differentiation of human pluripotent stem cells into hematopoietic endothelial cells (1) D-1 (Preparation stage one day before the start): After the confluence of human pluripotent stem cells reaches 80%, human pluripotent stem cells are digested into single cells using acutase and counted; 2×10 4 Human pluripotent stem cells were resuspended in 2 mL of mTeSR medium to obtain a cell suspension. The cell suspension was then seeded into one well of a six-well plate pre-coated with matrix gel. Finally, the human pluripotent stem cells were placed in a 37°C cell culture incubator containing 5% CO2 for adherent culture.
[0040] (2) D0 (starting day): The old culture medium was then aspirated by vacuum aspiration, and 2 mL of LDMMEM / F12 culture medium was added to each well of human pluripotent stem cells; then the human pluripotent stem cells were placed in a 37°C cell culture incubator containing 5% CO2 for six days to form hematopoietic endothelial cells.
[0041] (3) D6 (sixth day after the start date): Flow cytometry was used to detect CD34 expression. CD34 is a marker of hematopoietic endothelial cells, also known as hematopoietic endothelial cells or CD34+ hematopoietic endothelial cells. The results of flow cytometry detection of CD34 differentiation efficiency are as follows: Figure 7 As shown in the figure, the cells in the box are CD34 positive, accounting for 0.72%.
Claims
1. A method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells, characterized in that, Includes the following steps: a. Culture pluripotent stem cells until the pluripotent stem cell confluence reaches 50-80%; b. Digest pluripotent stem cells into single cells, resuspend the pluripotent stem cells in culture medium to obtain a cell suspension; c. Place the cell suspension into a cell culture plate and culture it in an adherent manner; d. After the culture in step c is completed, discard the culture medium described in step b, add hematopoietic differentiation culture medium to the cell culture plate, and induce culture to form CD34 positive hematopoietic endothelial cells; The hematopoietic differentiation culture medium consists of D / F12 medium and rhodioloside.
2. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, The pluripotent stem cells mentioned are human pluripotent stem cells.
3. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, In step a, the culture conditions are as follows: the culture is carried out in a cell culture incubator at 37°C containing 5% CO2, and the culture system is a feeder-free cell system. The specific operation is as follows: mTeSR medium is added to a cell culture plate covered with matrix gel, pluripotent stem cells are seeded into the mTeSR medium, and the mTeSR medium is changed every day.
4. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, Step b specifically includes: after the pluripotent stem cells have reached a confluence of 50-80%, digesting the pluripotent stem cells into single cells using acutase; then, distributing the pluripotent stem cells at a rate of 1×10⁻⁶ cells / cells. 4 The cells were resuspended in mTeSR medium at a concentration of 1 cell / mL to obtain a cell suspension.
5. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, The confluence of the pluripotent stem cells was 80%.
6. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, In step c, the cell culture plate is pre-coated with matrix gel, and the culture conditions are: cultured in a cell culture incubator at 37°C containing 5% CO2.
7. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, The concentration of rhodioloside is 10-100 μmol / L.
8. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 7, characterized in that, The concentration of rhodioloside is 100 μmol / L.
9. The method for inducing pluripotent stem cells to differentiate into CD34-positive hematopoietic endothelial cells according to claim 1, characterized in that, In step d, the method of discarding the culture medium mentioned in step b is to aspirate the culture medium by vacuum suction, and the conditions for induction culture are: cultured for 6 days in a cell culture incubator at 37°C containing 5% CO2.
10. CD34-positive hematopoietic endothelial cells prepared by the method according to any one of claims 1 to 9.