WO2023138680A1 - 诱导细胞分化的方法 - Google Patents

诱导细胞分化的方法 Download PDF

Info

Publication number
WO2023138680A1
WO2023138680A1 PCT/CN2023/073316 CN2023073316W WO2023138680A1 WO 2023138680 A1 WO2023138680 A1 WO 2023138680A1 CN 2023073316 W CN2023073316 W CN 2023073316W WO 2023138680 A1 WO2023138680 A1 WO 2023138680A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hematopoietic
stem cells
pluripotent stem
cell
Prior art date
Application number
PCT/CN2023/073316
Other languages
English (en)
French (fr)
Other versions
WO2023138680A9 (zh
WO2023138680A8 (zh
Inventor
李宗海
贾倩倩
Original Assignee
科济生物医药（上海）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 科济生物医药（上海）有限公司 filed Critical 科济生物医药（上海）有限公司
Publication of WO2023138680A1 publication Critical patent/WO2023138680A1/zh
Publication of WO2023138680A9 publication Critical patent/WO2023138680A9/zh
Publication of WO2023138680A8 publication Critical patent/WO2023138680A8/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present application relates to a method, a culture platform and a composition for inducing stem cell differentiation, in particular to a method, a culture platform and a composition for stem cells to differentiate into mesoderm, hematopoietic endothelium, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, T cells or NK cells.
  • HSCs Hematopoietic stem cells
  • HSCs are a type of adult stem cells present in hematopoietic tissues and blood, which have the ability of self-renewal and the potential to differentiate into all mature blood cells.
  • the occurrence and progression of most malignant blood diseases, such as acute myeloid leukemia and chronic myeloid leukemia, are directly or indirectly related to the abnormality of HSCs. Since its development in the 1960s, HSCs transplantation has become an important medical method for the treatment of various types of leukemia, aplastic anemia, ⁇ -thalassemia and other serious blood diseases and immune system diseases. It has also been applied to the treatment of certain solid tumors, such as lymphoma and small cell lung cancer.
  • HSCs for clinical application, namely bone marrow, peripheral blood and umbilical cord blood.
  • the extremely insufficient number of hematopoietic stem cells from natural sources is the main obstacle limiting its clinical application.
  • researchers have also developed methods for differentiation from embryonic stem cells or induced pluripotent stem cells to hematopoietic stem cells, in which the culture of mesoderm cells that undergo hematopoietic differentiation potential is required.
  • the widely recognized mesoderm differentiation methods with hematopoietic differentiation potential are limited, mainly including: 1) Activin A, BMP4, VEGF and bFGF; 2) CHIR99021, BMP4, Activin A (J Cell Physiol. 2020 August 10.), etc., all of which need to add multiple cytokines. Due to the high cost of cytokine preparation; and as a biologically active protein, it is easy to degrade; used in industry, the activity of different batches of cytokines varies greatly, is unstable, and needs to be added fresh; all of these lead to poor stability of the hematopoietic differentiation method containing cytokines, and difficulty in transformation and application.
  • this application establishes a safe, stable, efficient, and low-cost method for inducing stem cells to differentiate into mesoderm cells, hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, T cells or NK cells, a culture platform and a composition thereof.
  • the present application provides a novel method for rapid and efficient differentiation of the cells.
  • the specific technical scheme is as follows:
  • a method for inducing differentiation of stem cells by contacting pluripotent stem cells with a mesoderm differentiation-inducing agent at the mesoderm induction stage to produce mesoderm cells, the mesoderm differentiation-inducing agent comprising GSK-3 Inhibitor ⁇ Wnt pathway activator and compound A shown in formula I,
  • R1 is selected from:
  • the mesoderm differentiation inducer also includes a hypoxia-inducible factor (HIF) activator; preferably, the HIF activator is an HIF-1a activator.
  • HIF hypoxia-inducible factor
  • GSK-3 inhibitor/Wnt pathway activator is a small molecule compound.
  • X1 is selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, and halogen.
  • X2 is selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, and halogen.
  • X3 is selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, and halogen.
  • R2 is selected from:
  • the X4, X5, X6, or X7 are selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, or halogen, and X4, X5, X6, or X7 can be the same or different;
  • the R2 is selected from any of the following structures:
  • the HIF activator is a small molecule compound.
  • the CD56 or T gene of the mesoderm cells is positive.
  • GSK-3 inhibitor ⁇ Wnt pathway activator is selected from CHIR99021, SB216763, CHIR-98014, LY2090314, BIO-acetoxime, Alsterpaullone, CP21R7.
  • the HIF activator is a prolyl hydroxylase 2 inhibitor.
  • the HIF activator is selected from Roxadustat, IOX2, DMOG, PT-2385, Belzutifan, PX-478, LW6, Oltipraz, Daprodustat, BAY87-2243, KC7F2, Molidustat, PT2399, Amifostine, Enarodustat, Chlorogenic acid, ML228, Acriflavine, Vadadu stat, FG-2216, Tilorone dihydrochloride, Fraxinellone, 1,4-DPCA, MK-8617, Glucosamine hydrochloride, Hydralazine hydrochloride, AKBA.
  • the stem cell differentiation system does not contain serum or serum substitutes.
  • the stem cell differentiation system does not contain exogenously added cells
  • the exogenously added cells are trophoblast cells or co-culture cells.
  • the stem cell differentiation system is a monolayer differentiation system.
  • the stem cells are human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).
  • the method does not form endoderm cells or ectoderm cells.
  • the mesoderm differentiation inducer does not contain BMP4.
  • the planing density is not higher than 1*10 4 cells/cm 2 ; preferably, the planing density is not higher than 5000 cells/cm 2 .
  • the planing density is not lower than 1000 cells/cm 2 ; more preferably, the planing density is 1500-5000 cells/cm 2 .
  • the planing density is not lower than 1*10 4 cells/cm 2 ; preferably, the planing density is 1*10 4 cells/cm 2 -5*10 4 cells/cm 2 ; more preferably, the planing density is 3.7x10 4 -4.7x10 4 cells/cm 2 .
  • the plating density is 1000 cells/cm 2 -5*10 4 cells/cm 2 .
  • the added concentration of the GSK-3 inhibitor/Wnt pathway activator is 0.1-20 ⁇ M.
  • the added concentration of the compound represented by formula I is 0.1-20 ⁇ M.
  • the mesoderm cells are hematopoietic-related mesoderm cells.
  • the GSK-3 inhibitor/Wnt pathway activator is CHIR99021.
  • the mesoderm differentiation inducer does not contain Activin A.
  • the present application also provides the use of the mesoderm cells prepared by any of the aforementioned schemes for Further induce differentiation to prepare hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic stem progenitor cells, hematopoietic precursor cells and immune cells; preferably, the immune cells include T cells, NK cells, B cells, or macrophages.
  • Item 1 A method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines, characterized in that, in the hematopoietic endothelial cell induction stage, hematopoietic-related mesoderm cells derived from pluripotent stem cells are contacted with a composition comprising a high concentration of VEGF or a VEGF pathway activator to obtain hematopoietic endothelial cell populations.
  • Item 2 The method according to Item 1, wherein the composition comprises 30-200ng/ml VEGF, preferably 50-200ng/ml VEGF, more preferably 100-200ng/ml VEGF.
  • Item 3 A method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines, characterized in that, in the hematopoietic endothelial cell induction stage, hematopoietic-related mesoderm cells derived from pluripotent stem cells are contacted with a composition comprising a VEGF pathway activator to obtain hematopoietic endothelial cell populations.
  • Item 4 The method according to any one of Items 1-3, wherein the VEGF pathway activator includes shikonin derivatives; preferably, the shikonin derivatives include arnebin and its structural analogues.
  • Item 5 The method according to any one of items 1-4, wherein the VEGF pathway activator comprises Deoxyshikonin, Isoarnebin I or a combination thereof.
  • Item 6 The method according to any one of Items 1-5, wherein the composition further comprises a cAMP pathway activator.
  • Item 7 The method according to item 5 or 6, wherein the composition comprises 1-10 ⁇ M Deoxyshikonin; preferably, 1-6 ⁇ M Deoxyshikonin, more preferably, 2-6 ⁇ M Deoxyshikonin.
  • Item 8 The method according to Item 5 or 6, wherein the composition comprises 1-20 ⁇ M Isoarnebin I, preferably 5-15 ⁇ M Isoarnebin I, more preferably 8-15 ⁇ M Isoarnebin I.
  • Item 9 The method according to any one of Items 6-8, wherein the cAMP pathway activator includes Forskolin.
  • Item 10 The method according to Item 9, wherein the composition comprises 1-20 ⁇ M Forskolin; preferably, 2-10 ⁇ M Forskolin, more preferably, 2-4 ⁇ M Forskolin.
  • Item 11 The method according to any one of Items 1-10, wherein the composition further includes StemPro34.
  • Item 12 The method according to any one of Items 1-11, characterized in that, in the mesoderm cell guidance stage, iPSCs are contacted with another composition comprising Compound A to obtain the hematopoietic-related mesoderm cells; the other composition includes GSK-3 inhibitors ⁇ Wnt pathway activators, and compound A shown in formula I,
  • R1 is selected from:
  • Item 13 The method according to Item 12, wherein the other composition further comprises a hypoxia-inducible factor (HIF) activator; preferably, the HIF activator is an HIF-1a activator.
  • HIF hypoxia-inducible factor
  • Item 14 The method according to Item 12, wherein the GSK-3 inhibitor/Wnt pathway activator is a small molecule compound.
  • Item 15 The method according to Item 12, wherein in the compound represented by formula I, X1 is selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, and halogen.
  • Item 16 The method according to Item 12, wherein in the compound represented by formula I, X2 is selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, and halogen.
  • Item 17 The method according to Item 12, wherein in the compound represented by formula I, X3 is selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, and halogen.
  • Item 19 The method according to Item 18, wherein X4, X5, X6, or X7 are selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, or halogen, and X4, X5, X6, or X7 can be the same or different;
  • the R2 is selected from any of the following structures:
  • Item 20 The method according to Item 13, wherein the HIF activator is a small molecule compound.
  • Item 21 The method according to Item 12, wherein the mesoderm cells are positive for CD56 or T gene.
  • Item 22 The method according to Item 14, wherein the GSK-3 inhibitor/Wnt pathway activator is selected from CHIR99021, SB216763, CHIR-98014, LY2090314, BIO-acetoxime, Alsterpaullone, and CP21R7.
  • Item 23 The method according to Item 20, wherein the HIF activator is a prolyl hydroxylase 2 inhibitor.
  • Item 24 The method as described in Item 20, wherein the HIF activator is selected from Roxadustat, IOX2, DMOG, PT-2385, Belzutifan, PX-478, LW6, Oltipraz, Daprodustat, BAY87-2243, KC7F2, Molidustat, PT2399, Amifostine, Enarodustat, Chlorogenic acid, ML 228, Acriflavine, Vadadustat, FG-2216, Tilorone dihydrochloride, Fraxinellone, 1,4-DPCA, MK-8617, Glucosamine hydrochloride, Hydralazine hydrochloride, AKBA.
  • the HIF activator is selected from Roxadustat, IOX2, DMOG, PT-2385, Belzutifan, PX-478, LW6, Oltipraz, Daprodustat, BAY87-2243, KC7F2, Molidustat, PT2399, Amifostine, Enarodustat,
  • Item 25 The method according to any one of Items 1-24, wherein the differentiation of the pluripotent stem cells into a hematopoietic cell line under trophoblast-free conditions.
  • Item 26 The method according to any one of Items 1-25, wherein the differentiation of the pluripotent stem cells into a hematopoietic cell line is under a stromal-free condition or a stromal-containing condition.
  • Item 27 The method according to Item 26, wherein the matrix comprises matrigel, VTN-N, laminin-511, or laminin-521.
  • Item 28 The method according to any one of Items 1-27, wherein the composition and the other composition do not contain bFGF, BMP4 or Activin A.
  • Item 29 The method according to any one of Items 1-28, wherein the differentiation of the pluripotent stem cells into a hematopoietic cell line is a monolayer differentiation system.
  • Item 30 The method according to any one of Items 12-29, characterized in that the planing density is not higher than 1*10 4 cells/cm 2 ; preferably, the planing density is not higher than 5000 cells/cm 2 .
  • Item 31 The method according to any one of Items 1-30, characterized in that the planing density is not lower than 1000 cells/cm 2 ; more preferably, the planing density is 1500-5000 cells/cm 2 .
  • planing density is not less than 1*10 4 cells/cm 2 ; preferably, the planing density is 1*10 4 cells/cm 2 -5*10 4 cells/cm 2 ; more preferably, the planing density is 3.7x10 4 -4.7x10 4 cells/cm 2 .
  • Item 33 The method according to any one of Items 12-32, characterized in that the laying density is 1000 cells/cm 2 -5*10 4 cells/cm 2 .
  • Item 34 The method according to any one of Items 12-33, wherein the added concentration of the GSK-3 inhibitor/Wnt pathway activator is 0.1-20 ⁇ M.
  • Item 35 The method according to any one of Items 12-34, wherein the compound represented by formula I is added at a concentration of 0.1-20 ⁇ M.
  • Item 37 The method according to any one of Items 12-36, wherein the GSK-3 inhibitor/Wnt pathway activator is CHIR99021.
  • Item 38 The use of the blood endothelial cell population described in any one of Items 1-37, for preparing hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic precursor cells and immune cells through further guided differentiation; preferably, the immune cells include T cells, NK cells, B cells, or macrophages.
  • the method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines characterized in that, in the hematopoietic stem cell guiding stage, hematopoietic-related mesoderm cells or hematopoietic endothelial cells (HE) derived from pluripotent stem cells are contacted with composition I to obtain hematopoietic stem cells; said composition I comprises: Notch signaling pathway activator and TGF ⁇ receptor/ALK5 inhibitor.
  • the method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines characterized in that, in the hematopoietic stem cell induction stage
  • the first step is to contact the hematopoietic endothelial cells derived from pluripotent stem cells with composition II to obtain hematopoietic stem cells;
  • the composition II includes: Notch signaling pathway activator, TGF ⁇ receptor/ALK5 inhibitor and hematopoietic stem cell proliferation-promoting molecules.
  • Notch signaling pathway activator comprises Resveratrol and/or VPA.
  • TGF ⁇ receptor/ALK inhibitor includes SB431542, E-616452 and/or A83-01.
  • hematopoietic stem cell proliferation-promoting molecule comprises UM729, UM171, SR1 or a combination thereof.
  • the VEGF pathway activator includes shikonin derivatives; preferably, the shikonin derivatives include arnebin and structural analogs thereof.
  • VEGF pathway activator comprises Deoxyshikonin, Isoarnebin I or a combination thereof.
  • composition B further includes a cAMP pathway activator.
  • the added concentration of the Deoxyshikonin in the composition B is 1-5 ⁇ M
  • the added concentration of Isoarnebin I was 8-15 ⁇ M
  • the added concentration of Forskolin was 2-4 ⁇ M.
  • composition I, composition II or composition B further includes StemPro34.
  • composition I or composition II further includes ascorbic acid, SCF, IL-3, FLT-3L, GM-CSF, IL-6, TPO.
  • hematopoietic stem cells as described in any one of (1)-(22), for further inducing differentiation to prepare hematopoietic progenitor cells, hematopoietic precursor cells and immune cells; preferably, the immune cells comprise T cells, NK cells, B cells, or macrophages.
  • a method for inducing induced pluripotent stem cells to differentiate into hematopoietic cell lines characterized in that in the hematopoietic stem cell induction stage, hematopoietic-related mesoderm cells derived from iPSCs are contacted with composition III to obtain hematopoietic stem cells; said composition III comprises: high concentrations of VEGF and/or VEGF pathway activators, Notch signaling pathway activators, and TGF ⁇ receptor/ALK5 inhibitors.
  • composition III further includes a cAMP pathway activator.
  • TGF ⁇ receptor/ALK inhibitor includes SB431542, E-616452 and/or A83-01.
  • the VEGF pathway activator includes shikonin derivatives; preferably, the shikonin derivatives include arnebin and structural analogs thereof.
  • VEGF pathway activator comprises Deoxyshikonin, Isoarnebin I or a combination thereof.
  • composition III includes: VEGF, Deoxyshikonin, Isoarnebin I or a combination thereof, and also includes Resveratrol, Forskolin, and SB431542.
  • the method as described (24) is characterized by the adding concentration of the VEGF in the composition III is 100-200ng/ml or deoxyshikonin.
  • the addition concentration of Forskolin is 2-4 ⁇ m, and the adding concentration of SB431542 is 6-12 ⁇ m.
  • composition III also includes StemPro34, MCH5100, and/or SFEM II.
  • R1 is selected from:
  • composition A further includes a hypoxia-inducible factor (HIF) activator; preferably, the HIF activator is an HIF-1a activator.
  • HIF hypoxia-inducible factor
  • the R2 is selected from any of the following structures:
  • HIF activator is selected from Roxadustat, IOX2, DMOG, PT-2385, Belzutifan, PX-478, LW6, Oltipraz, Daprodustat, BAY87-2243, KC7F2, Molidustat, PT2399, Amifostine, Enarodustat, Chlorogenic acid, ML228, Acriflavine, Vadadustat, FG-2216, Tilorone dihydrochloride, Fraxinellone, 1,4-DPCA, MK-8617, Glucosamine hydrochloride, Hydralazine hydrochloride, AKBA.
  • the plating density of the pluripotent stem cells is not lower than 1*10 4 cells/cm 2 ; preferably, the plating density is 1*10 4 cells/cm2-5*10 4 cells/cm 2 ; more preferably, the plating density is 3.7x10 4 -4.7x10 4 cells/cm 2 .
  • (64) The use of mesoderm cells as described in any one of (40)-(63), for further inducing differentiation to prepare hematopoietic endothelium, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic precursor cells and immune cells; preferably, the immune cells comprise T cells, NK cells, B cells, or macrophages.
  • composition I comprises: Resveratrol and/or VPA; and SB431542 and/or A83-01.
  • composition II comprises: Resveratrol and/or VPA; SB431542 and/or A83-01; and UM729.
  • composition III comprises: high concentration VEGF or Deoxyshikonin or Isoarnebin, and also includes Resveratrol, SB431542 and Forskolin.
  • a method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines comprising: (i) contacting pluripotent stem cells with composition A comprising a GSK-3 inhibitor/Wnt pathway activator and compound A as described in (62) to obtain mesoderm cells; (ii) contacting mesoderm cells with composition B comprising a high concentration of VEGF or a VEGF pathway activator, and a cAMP pathway activator to obtain hematopoietic endothelium (HE); and (iii) contacting hematopoietic endothelial cells comprising Composition II of Notch signaling pathway activator, TGF ⁇ receptor/ALK5 inhibitor and hematopoietic stem cell pro-proliferation molecule to obtain hematopoietic stem cells.
  • composition A comprising a GSK-3 inhibitor/Wnt pathway activator and compound A as described in (62) to obtain mesoderm cells
  • composition B comprising a high concentration of VEGF or
  • a method for inducing differentiation of pluripotent stem cells into hematopoietic cell lines comprising: (i) contacting pluripotent stem cells with composition A comprising CHIR99021 and compound A as described in (62) to obtain mesoderm cells; (ii) contacting mesoderm cells with composition B containing high concentrations of VEGF or Deoxyshikonin or Isoarnebin, and Forskolin to obtain hematopoietic endothelial (HE); and (iii) making hematopoietic endothelial cells Hematopoietic stem cells were obtained by exposure to composition II comprising Resveratrol, SB431542 and UM729.
  • a method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines comprising: (i) contacting pluripotent stem cells with composition A comprising a GSK-3 inhibitor/Wnt pathway activator and compound A as described in (62) to obtain mesoderm cells; (ii) contacting mesoderm cells with composition III comprising high concentrations of VEGF and/or VEGF pathway activators, Notch signaling pathway activators, TGF ⁇ receptors/ALK5 inhibitors, and cAMP pathway activators to obtain production blood stem cells.
  • the method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines comprising: (i) contacting pluripotent stem cells with composition A comprising CHIR99021 and compound A as described in (62) to obtain mesoderm cells; (ii) contacting mesoderm cells with a composition comprising high concentrations of VEGF or Deoxyshikonin or Isoarnebin, Resveratrol, SB431542 and Composition III of Forskolin to obtain hematopoietic stem cells.
  • FIG. 1 An exemplary schematic diagram showing the three-stage process of induction of human induced pluripotent stem cells (hiPSCs) into hematopoietic stem cells (iHSCs).
  • hiPSCs human induced pluripotent stem cells
  • iHSCs hematopoietic stem cells
  • Figure 2 Schematic showing an exemplary two-stage process of hiPSC-to-iHSC-induced differentiation.
  • Figure 3A shows the expression of CD56 detected by flow cytometry when hiPSCs differentiated into hematopoietic mesoderm
  • Figure 3B shows the expression of T when hiPSCs differentiated into hematopoietic mesoderm detected by immunofluorescence.
  • the results showed that in the iHSC-A group that only added CHIR99021 and SB 4 without adding other cytokines (such as BMP4 and Activin A), T expression was detected in almost all cells, indicating that this differentiation system can achieve efficient directed differentiation of hematopoietic-related mesoderm.
  • Figure 4A shows the expression of TRA-1-85, SSEA4, and CD56 in mesoderm cells induced and differentiated by medium iHSC-A under the culture condition of VTN-N ( Figure 4A).
  • the results showed that under different ECM culture conditions, under the premise of successfully obtaining hematopoietic stem cells, the ubiquitous expression gene TRA-1-85 was stably and highly expressed during the directed differentiation of mesoderm, and the expression of the pluripotency marker gene SSEA4 gradually decreased, indicating that hiPSCs were gradually differentiating and losing pluripotency; CD56 presents a dynamic change process of decreasing first and then increasing, and showing high expression when reaching the mesoderm.
  • the high expression of CD56 indicates that this stage has entered the hematopoietic-related mesoderm stage. This shows that under a variety of ECM conditions (including VTN-N, matrigel, etc.), the directional differentiation of hematopoietic-related mesoderm can be achieved efficiently and stably, and hematopoietic-related mesoderm cells can be obtained, which shows the high efficiency of the iHSC culture platform for promoting differentiation.
  • Figure 5 shows the detection results of hematopoietic endothelial cells differentiated using iHSC culture platform 1:
  • Figure 5A shows that the differentiated CD34+ cell populations include CD73+ arteriovenous endothelial cells (AE/VE) and CD73- hematopoietic endothelial cells (HE).
  • AE/VE arteriovenous endothelial cells
  • HE CD73- hematopoietic endothelial cells
  • iHSC culture platform 1 successfully realized the directional differentiation of hematopoietic endothelial cells; when the positive rate of CD34, CD144, and CD31 reached about 20% to 30%, the positive rate of CD73 accounted for about 1/5; and the differentiated HE accounted for about 80%, and the arteriovenous endothelium (mainly arterial endothelium) accounted for about 20%. This indicates that the iHSC culture platform 1 achieves efficient directed differentiation of hematopoietic endothelium.
  • Figure 6 shows the detection results of iHSCs differentiated using iHSC culture platform 1 that includes iHSC-B including VEGF+Forskolin, Deoxyshikonin+Forskolin, and Isoarnebin I+Forskolin respectively:
  • Figure 6A, 6B, and 6C respectively The expression of CD34, CD45 and CD43 is shown.
  • the results show that the iHSC differentiation platform 1 can achieve nearly 100% CD43+CD45+ iHSC directional differentiation. This indicates that iHSC differentiation platform 1 achieves efficient and stable induction of hematopoietic stem cell differentiation.
  • Figure 7 shows the detection results of iHSCs differentiated using the iHSC culture platform 2 including iHSC-D (VEGF), iHSC-D (Deoxyshikonin), and iHSC-D (Isoarnebin I):
  • Figure 7A, 7B, and 7C respectively show the expression of CD34, CD45, and CD43.
  • the results show that the iHSC differentiation platform 2 can obtain nearly 100% directed differentiation of CD43+CD45+ iHSCs. This indicates that iHSC differentiation platform 2 achieves efficient and stable induction of hematopoietic stem cell differentiation.
  • Figures 8A and 8B show hiPSC high/low initial plating density directed differentiation to mesoderm cells, hematopoietic endothelium, iHSC early stage, iHSC late stage/iHPC early stage, and iHPC stage using iHSC culture platforms 1 and 2, respectively. Repeated three sets of experiments can obtain stable and consistent cell phenotype changes, indicating that iHSC culture platforms 1 and 2 have extremely high stability, which is of great significance for subsequent transformation applications to obtain stable and consistent high-quality cell products.
  • Figures 9A and B show the directed differentiation of iHSCs using iHSC platform 1 (Figure 9A) and the expression of CD34/CD45/CD43/CD31/CD38 in iHSCs under different ECM culture conditions ( Figure 9B).
  • Figures 9A and B show the directed differentiation of iHSCs using iHSC platform 1 ( Figure 9A) and the expression of CD34/CD45/CD43/CD31/CD38 in iHSCs under different ECM culture conditions ( Figure 9B).
  • the results showed that almost all the cells were CD43+CD45+, which indicated that under different ECM conditions, iHSC culture platform 1 could stably and efficiently differentiate into iHSCs.
  • FIG 10A, B and C show that iHSCs were differentiated using iHSC platform 2 under different basic medium and different ECM culture conditions: almost all cells were CD43+CD45+. This indicated that under different ECM culture conditions, iHSC platform 2 could differentiate iHSCs stably and efficiently.
  • FIG. 10B shows that under the culture condition of MyeloCult H5100 as the basic medium of iHSC-D, CD38+ myeloid cells accounted for about 20%, which shows that the iHSC obtained from this culture platform has a relatively obvious early myeloid differentiation tendency, so this culture platform can be used for myeloid differentiation and/or lymphoid differentiation, or preferably for myeloid differentiation.
  • Figures 10A and 10B respectively show that under the culture conditions of StemPro34 and StemSpan SFEM II as the basic medium of iHSC-D, the proportion of CD38+ myeloid cells is about 0% to 8%, which shows that this culture platform can be used for myeloid differentiation and/or lymphoid differentiation or lymphoid differentiation.
  • Figure 11 shows that the iHSCs obtained from the iHSC culture platforms 1 and 2 have totipotent hematopoietic differentiation ability using the CFU monoclonal formation assay.
  • Figure 12 shows that iHSCs obtained from iHSC culture platforms 1 and 2 have a tendency to spontaneously differentiate into myeloid-lymphoid lineages by using flow cytometry analysis, and also show that they have the potential of full-line hematopoietic differentiation.
  • Figure 13 shows that iHSCs obtained using iHSC culture platforms 1 and 2 can continue to differentiate into T cells after differentiation.
  • the present application relates to methods, culture platforms and compositions for differentiating stem cells to the hematopoietic lineage. More specifically, the present application provides a multi-stage culture platform, wherein iPSCs or iPSC-derived cells at different developmental stages can be induced to generate hematopoietic cell lineages ranging from mesoderm, hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic pluripotent progenitor cells to fully differentiated hematopoietic cells, the latter including T cells, B cells, NKT cells, NK cells and B cells.
  • the methods, culture platforms and compositions of the present application generate hematopoietic cell lineages in a scalable manner from naive iPSCs by avoiding the formation of EBs or aggregates.
  • the present application provides cell culture conditions, media, culture platforms and methods for culturing and differentiating stem cells to a hematopoietic fate.
  • the present application provides a combination of small molecules and cytokines for inducing differentiation of stem cells into hematopoietic-related mesoderm cells, hematopoietic endothelium, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, T cells, and NK cells and a culture platform thereof.
  • This application provides an inducing differentiation agent and a culture platform for stem cells to differentiate into hematopoietic-related mesoderm cells, hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, T cells, and NK cells without adding cytokines.
  • the present application provides methods and compositions for generating hematopoietic cell lineages from hematopoietic-associated mesoderm cells, hematopoietic endothelium, hematopoietic stem cells, and hematopoietic pluripotent progenitor cells from pluripotent stem cells, including hiPSCs, under serum-free/trophoblast-free conditions and in a scalable and monolayer culture platform without the need for EB formation.
  • Cells that can be differentiated according to the methods of the present application range from pluripotent stem cells to progenitor cells identified as specific terminally differentiated cells and transdifferentiated cells, cells of various lineages that are directly converted into hematopoietic cells without passing through pluripotent intermediates.
  • stem cell differentiation range from multipotent stem or progenitor cells to terminally differentiated stem cells, and all intermediate hematopoietic cell lineages.
  • the present application provides methods and compositions for differentiating and expanding hematopoietic cell lineages from pluripotent stem cells in monolayer culture, comprising contacting pluripotent stem cells with a GSK3 ⁇ inhibitor and the small molecule SB4 to obtain and expand mesoderm cells without forming embryoid bodies from pluripotent stem cells.
  • the mesoderm cells are then contacted with high concentrations of VEGF and/or VEGF pathway activators and optionally cAMP pathway activators to obtain hematopoietic endothelium without formation of embryoid bodies.
  • the present application provides methods and compositions for differentiating and expanding hematopoietic cell lineages from pluripotent stem cells in monolayer culture, comprising contacting pluripotent stem cells with a GSK3 ⁇ inhibitor and the small molecule SB4 to obtain and expand mesoderm cells without forming embryoid bodies from pluripotent stem cells.
  • the mesoderm cells are then contacted with Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optionally cAMP pathway activators, and optionally high concentrations of VEGF and/or VEGF pathway activators to obtain and expand hematopoietic stem cells without forming embryoid bodies.
  • the method presented here for obtaining hematopoietic cell lineages using monolayer differentiation is superior to EB-mediated differentiation of pluripotent stem cells because EB formation leads to heterogeneous cell differentiation and inefficient cell expansion.
  • This application provides a small molecule combination inducing differentiation agent without adding cytokines, and establishes a monolayer culture platform that is conducive to differentiation into hematopoietic-related mesoderm, hematopoietic endothelium, hematopoietic stem cells, and hematopoietic pluripotent progenitor cells, leading to the derivation of hematopoietic stem cells, hematopoietic pluripotent progenitor cells and their differentiated progeny such as T, B, NKT, and NK cells.
  • our small-molecule combinatorial inducer and monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion to deliver therapeutically relevant quantities of pluripotent stem cell-derived hematopoietic cell lineages for a variety of therapeutic applications.
  • the present application discloses the use of small molecule combinations to induce differentiation agents and monolayer culture methods to obtain functional hematopoietic cell lineages, which are capable of in vitro differentiation, ex vivo regulation, and long-term hematopoietic self-renewal, hematopoietic reconstitution and hematopoietic transplantation in vivo.
  • the present application provides a culture platform for obtaining hematopoietic-related mesoderm cells derived from pluripotent stem cells, which comprises: Group I: (i) a medium comprising a GSK3 ⁇ inhibitor and a small molecule SB4, and the medium is suitable for differentiating and expanding hematopoietic-related mesoderm cells from pluripotent stem cells.
  • the application provides a culture platform for obtaining hematopoietic cell lineage derived from pluripotent stem cells, which comprises: Group II: (i) culture medium, which includes GSK3 ⁇ inhibitors and small molecule SB4, which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which contains high concentrations of VEGF and/or VEGF pathway activators and optional cAMP pathway activators, and which is suitable for differentiating and expanding hematopoietic endothelial cells from mesoderm cells; (iii) culture medium, which comprises Notch signaling pathway activation and TGF ⁇ receptor/ALK inhibitors, and optional hematopoietic stem cell pro-proliferation molecules, the medium is suitable for differentiating and expanding hematopoietic stem cells from hematopoietic endothelium.
  • the application provides a culture platform for obtaining hematopoietic cell lineage derived from pluripotent stem cells, which comprises: Group III: (i) culture medium, which contains GSK3 ⁇ inhibitors and small molecule SB4, which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) culture medium, which contains Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optional cAMP pathway activators, and optional high-concentration VEGF and/or VEGF pathway activators, and this medium is suitable for mesodermal cells Differentiate and expand hematopoietic stem cells.
  • the present application provides a culture platform for obtaining hematopoietic stem cells derived from pluripotent stem cells, which comprises: Group II: (i) medium, which comprises GSK3 ⁇ inhibitors and small molecule SB4, and which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which contains high concentrations of VEGF and/or VEGF pathway activators and optional cAMP pathway activators, and which is suitable for differentiating and expanding hematopoietic endothelial cells from mesoderm cells; (iii) medium, which comprises Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, and optional hematopoietic stem cell pro-proliferation molecules, the culture Suitable for differentiation and expansion of hematopoietic stem cells from hematopoietic endothelium.
  • the present application provides a culture platform for obtaining hematopoietic stem cells derived from pluripotent stem cells, which comprises: Group III: (i) medium, which includes GSK3 ⁇ inhibitors and small molecule SB4, which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which contains Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optional cAMP pathway activators, and optional high-concentration VEGF and/or VEGF pathway activators, and this medium is suitable for differentiation and expansion from mesoderm cells Expand hematopoietic stem cells.
  • Group III group III: (i) medium, which includes GSK3 ⁇ inhibitors and small molecule SB4, which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which contains Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optional cAMP pathway activators, and optional high-concentration VEGF and/or
  • the application provides a culture platform for obtaining hematopoietic pluripotent progenitor cells derived from pluripotent stem cells, which comprises: Group II: (i) medium, which includes GSK3 ⁇ inhibitors and small molecule SB4, which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which contains high concentrations of VEGF and/or VEGF pathway activators and optional cAMP pathway activators, and this medium is suitable for differentiation and expansion of hematopoietic endothelial cells from mesoderm cells; (iii) medium, which comprises Notch signaling pathway Activators and TGF ⁇ receptor/ALK inhibitors, and optionally hematopoietic stem cell pro-proliferative molecules, the medium is suitable for differentiating and expanding hematopoietic stem cells from hematopoietic endothelium.
  • medium which includes GSK3 ⁇ inhibitors and small molecule SB4, which is suitable for differentiating and expanding me
  • the present application provides a culture platform for obtaining hematopoietic pluripotent progenitor cells derived from pluripotent stem cells, which comprises: Group III: (i) culture medium, which comprises GSK3 ⁇ inhibitors and small molecule SB4, and which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which comprises Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optional cAMP pathway activators, and optional high-concentration VEGF and/or VEGF pathway activators, and this medium is suitable for differentiation and expansion of mesoderm cells from mesoderm Cell differentiation and expansion of hematopoietic stem cells.
  • Group III comprises: (i) culture medium, which comprises GSK3 ⁇ inhibitors and small molecule SB4, and which is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) medium, which comprises Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optional
  • the pluripotent stem cells of the aforementioned culture platforms are iPSCs.
  • the iPSCs are naive iPSCs.
  • Groups I, II, and III of the above-mentioned culture platform do not contain cytokine components such as BMP4, Activin A, bFGF, LIF, and serum components such as FBS and KOSR.
  • the above culture platform it further comprises an additional medium, which is suitable for seeding and expanding pluripotent stem cells.
  • the (i) culture medium of Group I, II or III further comprises: one or more selected from DMEM/F12, Neurobasal, N2, B27 and ⁇ -Me.
  • the (ii) culture medium of group II further comprises: one or more selected from StemPro34, Glutamine.
  • the (iii) culture medium of group II further comprises: one or more selected from StemPro34, Glutamine, SCF, GM-CSF, IL-3, IL-6, FLT-3L, TPO, ascorbic acid.
  • the (ii) medium of Group III further comprises: one or more selected from StemPro34, Glutamine, ascorbic acid; or one or more selected from MCH5100, ascorbic acid; or one or more selected from SFEM II, ascorbic acid.
  • the present application provides a culture platform for obtaining hematopoietic endothelial cells derived from pluripotent stem cells, which comprises: Group IV: (i) a culture medium comprising a high concentration of VEGF and/or a VEGF pathway activator and an optional cAMP pathway activator, the medium being suitable for differentiating and expanding hematopoietic endothelial cells from mesoderm cells.
  • Group IV of the culture platform above is free of GSK3 ⁇ inhibitors, Activin A, bFGF, wherein the medium is suitable for seeding and expanding mesoderm cells.
  • the (i) medium of group IV further comprises additional medium: one or more selected from StemPro34, Glutamine.
  • the application provides a culture platform for obtaining hematopoietic stem cells derived from pluripotent stem cells, which includes: Group V: (i) a medium comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, and optional hematopoietic stem cell proliferation-promoting molecules, the medium is suitable for differentiating and expanding hematopoietic stem cells from hematopoietic endothelium.
  • Group V of the culture platform above is free of GSK3 ⁇ inhibitors, VEGF pathway activators, cAMP pathway activators, BMP pathway activators, wherein the medium is suitable for seeding and expanding hematopoietic endothelium.
  • the (i) culture medium of group V further comprises: one or more selected from StemPro34, Glutamine, SCF, GM-CSF, IL-3, IL-6, FLT-3L, TPO, ascorbic acid.
  • the present application provides a culture platform for obtaining hematopoietic stem cells derived from pluripotent stem cells, which comprises: Group VI: (i) medium, which comprises Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optional cAMP pathway activators, and optional high-concentration VEGF and/or VEGF pathway activators, and the medium is suitable for differentiation and expansion of hematopoietic stem cells from mesoderm cells.
  • Group VI of the culture platform above is free of bFGF, BMP pathway activators, GSK3 ⁇ inhibitors, wherein the medium is suitable for seeding and expanding mesoderm cells.
  • the (i) medium of group VI further comprises an additional medium: one or more selected from StemPro34, Glutamine, ascorbic acid; or one or more selected from MCH5100, ascorbic acid; or one or more selected from SFEM II, ascorbic acid.
  • the culture medium of each group may also optionally contain a hypoxia-inducible factor (HIF) activator.
  • HIF hypoxia-inducible factor
  • the present application also provides a composition for differentiating and expanding a pluripotent stem cell-derived hematopoietic cell lineage, which comprises one or more of the following groups I, II, III, IV, V or VI.
  • Group I (i) media comprising a GSK3 ⁇ inhibitor and the small molecule SB4; and iPSCs and suitable for differentiation and expansion of hematopoietic-related mesoderm cells from pluripotent stem cells.
  • Group II (i) a culture medium comprising a GSK3 ⁇ inhibitor and the small molecule SB4; and iPSCs, a medium suitable for differentiating and expanding mesodermal cells from pluripotent stem cells; (ii) a medium comprising high concentrations of VEGF and/or VEGF pathway activators and optionally a cAMP pathway activator; and mesoderm cells, a medium suitable for differentiating and expanding hematopoietic endothelium from mesoderm cells; Proliferative molecules of hematopoietic stem cells, and hematopoietic endothelium, the medium is suitable for differentiating and expanding hematopoietic stem cells from hematopoietic endothelium.
  • Group III (i) a medium comprising a GSK3 ⁇ inhibitor and a small molecule SB4, and iPSCs suitable for differentiating and expanding mesoderm cells from pluripotent stem cells; (ii) a medium comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optionally cAMP pathway activators, and optionally high concentrations of VEGF and/or VEGF pathway activators, and mesoderm cells, which medium is suitable for differentiating and expanding hematopoietic stem cells from mesoderm cells.
  • Group IV (i) a culture medium comprising high concentrations of VEGF and/or VEGF pathway activators and optionally cAMP pathway activators, and mesodermal cells, the medium is suitable for differentiation and expansion of hematopoietic endothelium from pluripotent stem cell-derived mesoderm cells.
  • Group V (i) a medium comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, and optional hematopoietic stem cell pro-proliferation molecules, and hematopoietic endothelium, the medium is suitable for differentiation and expansion of hematopoietic stem cells from pluripotent stem cell-derived hematopoietic endothelium.
  • Group VI (i) a medium comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK5 inhibitors, and optionally cAMP pathway activators, and optionally high concentrations of VEGF and/or VEGF pathway activators, and mesoderm cells, the medium being suitable for differentiation and expansion of hematopoietic stem cells from pluripotent stem cell-derived mesoderm cells.
  • the pluripotent stem cell is an iPSC.
  • the iPSCs are naive iPSCs.
  • the (i) medium of Group I, II, or III comprises an additional component: one or more selected from the group consisting of DMEM/F12, Neurobasal, N2, B27, and ⁇ -Me.
  • the culture medium of Group II (ii) and Group IV (i) comprises additional components: one or more selected from StemPro34, Glutamine.
  • the (iii) culture medium of group II comprises additional components: one or more selected from StemPro34, Glutamine, SCF, GM-CSF, IL-3, IL-6, FLT-3L, TPO, ascorbic acid.
  • the (ii) of Group III, (i) of Group V, and (i) culture medium of Group VI comprise accessory components: one or more selected from StemPro34, Glutamine, ascorbic acid; or one or more selected from MCH5100, ascorbic acid; or one or more selected from SFEM II, ascorbic acid.
  • groups (I), (II), (III), (IV), (V), (VI) further comprise: (i) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; and one or more Notch pathway activators; and pluripotent stem cell-derived T progenitor cells, wherein the medium is free of BMPs activator, and suitable for differentiating T progenitor cells derived from pluripotent stem cells into T cells, or (ii) a medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6; one or more Notch pathway activators; and iHSC derived from pluripotent stem cells, wherein the medium is suitable
  • the present application also provides a method for guiding pluripotent stem cells to differentiate into mesoderm cells, comprising: (i) contacting pluripotent stem cells with a composition comprising a GSK3 ⁇ inhibitor and a small molecule SB4 to initiate differentiation and expansion from pluripotent stem cells to hematopoietic-related mesoderm cells.
  • a method for directing the differentiation of pluripotent stem cells into a hematopoietic cell lineage comprising: (i) contacting pluripotent stem cells with a composition comprising a GSK3 ⁇ inhibitor and a small molecule SB4 to initiate differentiation and expansion from pluripotent stem cells to mesoderm cells; (ii) contacting mesoderm cells with a composition comprising a high concentration of VEGF and/or a VEGF pathway activator and optionally an activator of the cAMP pathway to initiate differentiation and expansion from mesoderm cells to hematopoietic endothelium; The blood endothelium is exposed to a composition comprising a Notch signaling pathway activator and a TGF ⁇ receptor/ALK inhibitor, and optionally a hematopoietic stem cell pro-proliferative molecule, to initiate differentiation and expansion of the hematopoietic stem cells from the hematopoietic endothelium.
  • a method for directing the differentiation of pluripotent stem cells into a hematopoietic cell lineage comprising: (i) contacting pluripotent stem cells with a composition comprising a GSK3 ⁇ inhibitor and a small molecule SB4 to initiate differentiation and expansion from pluripotent stem cells to mesoderm cells; and (ii) contacting the mesoderm cells with a composition comprising a Notch signaling pathway activator and a TGF ⁇ receptor/ALK5 inhibitor, and optionally a cAMP pathway activator, and optionally a high concentration of VEGF and/or a VEGF pathway activator, to Initiates differentiation and expansion of mesoderm cells into hematopoietic stem cells.
  • a method for inducing differentiation of pluripotent stem cells into hematopoietic endothelium comprising: (i) contacting pluripotent stem cell-derived mesoderm cells with a composition comprising a high concentration of VEGF or a VEGF pathway activator and optionally a cAMP pathway activator to initiate differentiation and expansion from the mesoderm cells to hematopoietic endothelium.
  • a method for guiding pluripotent stem cells to differentiate into hematopoietic stem cells comprising: (i) contacting hematopoietic endothelium derived from pluripotent stem cells with a composition comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, and optional hematopoietic stem cell proliferation-promoting molecules, to initiate differentiation and expansion from hematopoietic endothelium to hematopoietic stem cells.
  • a method for guiding pluripotent stem cells to differentiate into hematopoietic stem cells comprising: (i) making pluripotent stem cell-derived mesoderm cells contact a composition comprising a Notch signaling pathway activator and a TGF ⁇ receptor/ALK5 inhibitor, and an optional cAMP pathway activator, and an optional high concentration of VEGF or a VEGF pathway activator, to initiate differentiation and expansion from mesoderm cells to hematopoietic stem cells.
  • pluripotent stem cells In the above method for inducing the differentiation of pluripotent stem cells into hematopoietic cell lineages, the differentiation of pluripotent stem cells into hematopoietic cell lineages does not produce embryoid bodies and is in the form of monolayer culture.
  • pluripotent stem cells, mesoderm cells, hematopoietic endothelium, hematopoietic stem cells, and/or hematopoietic pluripotent progenitor cells are subjected to hypoxic conditions, or small molecules are added to activate cellular hypoxic pathways.
  • the pluripotent stem cells are iPSCs. In some instances, the iPSCs are naive iPSCs.
  • the obtained mesoderm cells are Brachyury+CD56+.
  • the obtained hematopoietic endothelium is CD31+CD34+CD184+.
  • the obtained hematopoietic endothelium is CD31+CD144+CD34+CD184+.
  • the obtained hematopoietic endothelium is CD144+CD34+CD184+.
  • the obtained hematopoietic stem cells are CD34+CD43+CD45+ or hematopoietic stem cells are CD43+CD45+.
  • the obtained hematopoietic pluripotent progenitor cells are CD34+CD43+CD45+ or hematopoietic pluripotent progenitor cells are CD43+CD45+.
  • the present application provides a method for generating hematopoietic stem cells derived from pluripotent stem cells, comprising: (i) contacting iPSCs with a composition comprising a GSK3 ⁇ inhibitor and a small molecule SB4 to initiate differentiation and expansion from pluripotent stem cells to mesoderm cells; (ii) contacting mesoderm cells with a composition comprising a high concentration of VEGF and/or a VEGF pathway activator and an optional cAMP pathway activator to initiate differentiation and expansion from mesoderm cells to hematopoietic endothelium; The endothelium is contacted with a composition comprising a Notch signaling pathway activator and a TGF ⁇ receptor/ALK inhibitor, and optionally a hematopoietic stem cell pro-proliferative molecule, to initiate differentiation and expansion from the hematopoietic endothelium to hematopoietic stem cells; and optionally, subjecting the seeded pluripot
  • the above method of generating pluripotent stem cell-derived hematopoietic stem cells further comprises: contacting the iPSCs with a maintenance medium comprising a ROCK inhibitor to seed and expand the iPSCs; and/or wherein the iPSCs are naive iPSCs.
  • the methods described above for differentiating iPSCs into hematopoietic stem cells do not produce embryoid bodies and are monolayer cultured.
  • the present application provides a method for generating hematopoietic stem cells derived from pluripotent stem cells, comprising: (i) contacting iPSCs with a composition comprising a GSK3 ⁇ inhibitor and a small molecule SB4 to initiate differentiation and expansion from pluripotent stem cells to mesoderm cells; (ii) contacting the mesoderm cells with a composition comprising a Notch signaling pathway activator and a TGF ⁇ receptor/ALK5 inhibitor, and optionally a cAMP pathway activator, and optionally a high concentration of VEGF or a VEGF pathway activator to initiate differentiation from the iPSC Differentiation and expansion of germ layer cells to hematopoietic stem cells; and optionally subjecting seeded pluripotent stem cells, mesoderm cells to a low oxygen tension of about 2% to about 10%.
  • the above-mentioned generation of hematopoietic stem cells derived from pluripotent stem cells The cell method further comprises: contacting the iPSCs with a maintenance medium comprising a ROCK inhibitor to seed and expand the iPSCs; and/or wherein the iPSCs are naive iPSCs.
  • the methods described above for differentiating iPSCs into hematopoietic stem cells do not produce embryoid bodies and are in monolayer culture.
  • the present application provides a composition or a plurality of cell populations comprising: one or more cell populations produced from the culture platform disclosed herein, the methods described herein: pluripotent stem cell-derived (i) Brachyury+CD56+ mesoderm cells capable of differentiating into hematopoietic endothelium, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, T progenitor cells, NK progenitor cells, T cells and NK cells; (ii) CD31+ (or CD144)+CD34+CD73-CD4 3 - CD184+ hematopoietic endothelium; (iii) CD43+CD45+ hematopoietic stem cells; (iv) T progenitors, wherein T progenitors are CD34+CD7+; (v) T cells, wherein T cells are CD4+ or CD8+; (vi) NK progenitors, wherein NK progenitors are
  • the present application provides methods for promoting hematopoietic self-renewal, reconstitution or transplantation using one or more cell populations, cell lines or clones produced by the disclosed methods: pluripotent stem cell derived (i) Brachyury+CD56+ mesoderm cells capable of differentiating into hematopoietic endothelial cells, hematopoietic stem cells, T progenitor cells, NK progenitor cells, T cells and NK cells; (ii) CD31+ (or CD144)+CD34+CD73-CD43-CD184+ cells (iii) CD43+CD45+ hematopoietic stem cells; (iv) T progenitor cells, wherein the T progenitor cells are CD34+CD7+; (v) T cells, wherein the T cells are CD4+ or CD8+; (vi) NK progenitor cells, wherein the NK progenitor cells are CD56+CD7+CD161+; and (vii)
  • the present application provides methods and compositions that enable the direct differentiation of pluripotent stem cell monolayers without generating embryoid bodies from pluripotent stem cells, thereby enabling the differentiation and expansion of mesoderm cells, hematopoietic endothelial cells, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, wherein other hematopoietic cell lineages can be obtained in a scalable, reliable format at very high efficiency levels.
  • the high concentration refers to a concentration that can reach a concentration of 30 ng/mL or more, such as a concentration of 30-500 ng/mL, such as a concentration of 10-500 ng/mL, such as a concentration of 30-30 ng/mL, for example, the concentration can be 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 8 0ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, 200ng/mL, 210ng/mL, 220ng/mL, 230ng/mL, 240ng/mL, 250
  • hypoxic condition refers to a low oxygen tension of about 2% to about 10%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% oxygen concentration conditions.
  • the term “about” or “approximately” refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In some instances, the term “about” or “approximately” refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.
  • the term “substantially” or “substantially” refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.
  • the term “substantially the same” or “substantially the same” refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “substantially free” and “substantially free” are used interchangeably and when used to describe a composition such as a cell population or a culture medium, refers to a composition that is free of a specified substance or its source, e.g., 95% free, 96% free, 97% free, 98% free, 99% free of a specified substance or its source, or is undetectable as measured by conventional means.
  • the term “free” or “substantially free” of an ingredient or substance in a composition also means that the ingredient or substance (1) is not included in the composition at any concentration, or (2) has an inert function in the composition, but is present in a low concentration. A similar meaning applies to the term “absent” when referring to the absence of a particular substance of the composition or its source.
  • the term “evaluable” refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length range that is readily detectable by one or more standard methods.
  • the terms “not appreciable” and “not appreciable” and equivalents refer to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or range of length that is not readily detectable or undetectable by standard methods. In some instances, an event is not evaluable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less in time.
  • T gene is a tissue-specific transcription factor encoded by Brachury.
  • human pluripotent stem cells including human induced pluripotent stem cells and human embryonic stem cells
  • monolayer directed hematopoietic-related mesoderm differentiation system or multilayer differentiation system In the stem cell differentiation system, human pluripotent stem cells (including human induced pluripotent stem cells and human embryonic stem cells) monolayer directed hematopoietic-related mesoderm differentiation system or multilayer differentiation system.
  • mammals refers to one of three germ layers that arise during early embryogenesis and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, bone, and other supportive and connective tissues.
  • hematopoietic cell lineage and “hematopoietic cell lineage” refer to all types of hematopoietic-related cell groups involved in the process of hematopoietic development or hematopoietic differentiation, including and not limited to hematopoietic endothelium, hematopoietic stem cells, hematopoietic pluripotent progenitor cells, hematopoietic progenitor cells (lymphoid progenitor cells and myeloid progenitor cells, etc.), double negative T progenitor cells, double positive T progenitor cells, T progenitor cells, NK progenitor cells, T cells and NK cells.
  • EHT endothelial to hematopoietic transition
  • HECs early hematopoietic endothelial cells
  • HSPCs hematopoietic stem cells
  • hemogenic endothelium or “hemogenic endothelium” (HE, Hemogenic Endothelium) or “pluripotent stem cell-derived permanent hematopoietic endothelium” (iHE) refer to the subpopulation of endothelial cells that give rise to hematopoietic stem cells and hematopoietic progenitor cells in a process known as the endothelial-hematopoietic transition.
  • Hemopoietic stem cell refers to an immature cell, which is the origin of all hematopoietic cells and immune cells. It can not only differentiate into red blood cells, white blood cells and platelets, but also differentiate into cells of various tissues and organs across the system. It is capable of producing mature myeloid and lymphoid cell types, including T cells, natural killer cells and B cells.
  • hematopoietic stem cells include a pool of hematopoietic stem and progenitor cells (HSPCs).
  • a pool of hematopoietic stem and progenitor cells can include three types of cells: long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSPCs), and hematopoietic multipotent progenitor cells (MPPs).
  • LT-HSCs long-term hematopoietic stem cells
  • ST-HSPCs short-term hematopoietic stem cells
  • MPPs hematopoietic multipotent progenitor cells
  • HSC Lin-CD34+CD43+CD45+CD38-CD45RA-CD90+CD49f+
  • LT-HSC CD34-, CD38-, SCA-1+, Thy1.1+/lo, C-kit+, lin-, CD135-, Slamf1/CD150+
  • ST-HSC CD 34+, CD38+, SCA-1+, Thy1.1+/lo, C-kit+, lin-, CD135-
  • MPP CD34+CD43+CD45+CD38-CD45RA-CD90-CD49f-.
  • hematopoietic multipotent progenitor cell is an immature cell. Hematopoietic progenitor cells are mainly developed and differentiated from hematopoietic stem cells. Compared with hematopoietic stem cells, they also have the potential to develop and differentiate into full-line hematopoietic cells, and they can also differentiate into bone marrow. However, the main difference from hematopoietic stem cells is that they no longer have the ability to self-renew.
  • hematopoietic progenitor cells also known as multipotent hematopoietic progenitor cells
  • HPCs hematopoietic progenitor cells
  • multipotent hematopoietic progenitor cells refers to hematopoietic stem cells that proliferate and differentiate into various types of blood cell progenitor cells under the regulation of certain microenvironment and certain factors. It is also a kind of primitive cell with proliferation ability, but it has lost the ability of multidirectional differentiation and can only proliferate and differentiate toward one or several blood cell lines, so it is also called committed stem cell (committed stem cell).
  • reprogramming or “dedifferentiation” or “increasing the potential of a cell” or “increasing developmental potential” refer to methods of increasing the potential of a cell or dedifferentiating a cell to a less differentiated state.
  • a cell with increased cellular potential has more developmental plasticity (ie, differentiates into more cell types) than the same cell in a non-reprogrammed state.
  • a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.
  • differentiation is the process by which a non-specific or less specific cell acquires the characteristics of a specific cell such as a blood cell or a muscle cell.
  • Differentiated or differentiation-inducing cells are cells that occupy a more specific position in a cell lineage.
  • the term "determined” when applied to the differentiation process refers to a cell that has progressed to a point in the differentiation pathway where it would normally go on to differentiate into a particular cell type or subpopulation of cell types and would not normally be able to differentiate into a different cell class type or reversion to a poorly differentiated cell type.
  • Differentiation marker gene refers to a gene whose expression is indicative of cellular differentiation occurring within a cell, such as a pluripotent cell.
  • Differentiation marker genes include but are not limited to the following genes: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH1 1.
  • ZIC4 GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D, FOXG1, LEFTY1, TUJ1, T gene (Brachyury), ZIC1, GATA1, GATA2, HDAC4, HDAC5, HDAC7, HDAC9, NOTCH1, NOTCH2, NOTCH4, PAX5, RBPJ, RUNX1, STAT1, and STAT3.
  • differential marker gene profile or “differentiation gene profile”, “differentiation gene expression profile”, “differentiation gene expression signature”, “differentiation gene expression panel”, or “differentiation gene signature” refers to the expression or expression level of multiple differentiation marker genes.
  • the term "potency” refers to the sum of all developmental options accessible to a cell (ie, developmental potential).
  • the continuum of cellular potential includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells.
  • pluripotent refers to the ability of a cell to form all lineages of an organism or body (ie, an embryonic body).
  • embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm.
  • Pluripotency is a continuum of developmental potential, ranging from incomplete or partially pluripotent cells that cannot give rise to complete organs (for example, ectodermal stem cells or EpiSCs) to primitive, more pluripotent cells that can give rise to complete organs (for example, embryonic stem cells).
  • induced pluripotent stem cells or “induced pluripotent stem cells”, or iPSCs, means that stem cells arise from differentiated adult/neonatal or fetal cells that have been induced or altered, i.e., cells reprogrammed into tissues capable of differentiating into all three germ or dermal layers: mesoderm, endoderm and ectoderm.
  • the iPSCs produced do not refer to cells found in nature.
  • fibroblasts or blood cells are isolated from blood samples, activated, and retrovirally reprogrammed into iPSCs.
  • fibroblasts or blood cells are isolated from human blood samples, activated, and retrovirally reprogrammed into hiPSCs.
  • T cells were isolated from human blood samples, activated, and retrovirally reprogrammed into hiPSCs.
  • embryonic stem cells refers to naturally occurring pluripotent stem cells of the inner cell mass of embryonic blastocysts. Embryonic stem cells are pluripotent and during development give rise to all derivatives of the three main germ layers: ectoderm, endoderm and mesoderm. They do not contribute to the extraembryonic membrane or the placenta, i.e. are not totipotent.
  • multipotent stem cell refers to a cell that has the developmental potential to differentiate into one or more germ layers (ectoderm, mesoderm and endoderm), but not all three. Therefore, multipotent cells may also be referred to as “partially differentiated cells”. Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. “Multipotent” means that a cell may form many types of cells in a given lineage, but not cells of other lineages.
  • EBs embryoid bodies
  • naive hiPSCs are seeded as a monolayer in maintenance medium and allowed to expand until a plating density of about 25% and above is reached. In some examples, naive hiPSCs are seeded as monolayer cultures in maintenance medium and allowed to expand until reaching a plating density within about 70%. In some examples, the initial hiPSCs are inoculated in a monolayer culture system at a plating density of not less than about 1500-5000 cells/cm 2 in a maintenance medium.
  • initial hiPSCs are seeded in a monolayer culture system in a maintenance medium at a plating density of about 3.7x10 4 -4.7x10 4 cells/cm 2 and above. In some examples, initial hiPSCs are seeded in a monolayer culture system in a maintenance medium at a plating density not higher than about 2.5x10 5 cells/cm 2 . In some examples, the primary hiPSCs are seeded in a monolayer culture system in a maintenance medium at a plating density of about 2.5x10 4 -1x10 5 cells/cm 2 .
  • initial hiPSCs are seeded in a monolayer culture system in a maintenance medium at a plating density of about 2.5x10 4 -3x10 4 cells/cm 2 .
  • initial hiPSCs are seeded in a monolayer culture system in a maintenance medium at a plating density of about 8x10 4 -1x10 5 cells/cm 2 .
  • Pluripotency can be determined, in part, by evaluating the pluripotency characteristics of cells.
  • Pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) unlimited self-renewal potential, (iii) expression of the following pluripotent stem cell markers including, but not limited to: SSEA1 (mouse only), SSEA3/4, SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD 140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and/or CD50; (iv) ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation consisting of three somatic lineages; and (vi) embryoid body formation consisting of cells from the three somatic lineages.
  • pluripotent stem cell morphology refers to the typical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape with a high nucleoplasmic ratio, the prominent presence of nucleoli and typical intercellular spacing.
  • adherent cells refers to the attachment of cells to a vessel, eg, to a sterile plastic (or coated plastic) cell culture dish or flask, in the presence of an appropriate medium. Certain types of cells cannot persist or grow in culture unless they adhere to the cell culture vessel. Certain classes of cells (“non-adherent cells”) do not require attachment to maintain and/or proliferate in culture.
  • extracellular matrix refers to the substances and corresponding structures formed by a complex network of various macromolecules surrounding cells in a multicellular organism.
  • the extracellular matrix is mainly composed of 5 types of substances, namely collagen, non-collagen, elastin, proteoglycan and aminoglycan, which are mainly divided into basement membrane and interstitial matrix according to the distribution site.
  • the extracellular matrix used in cell culture is mostly composed of extracts or chemically synthesized or purified by biological expression, which forms a relatively stable extracellular support substance.
  • culture refers to the maintenance, growth and/or differentiation of cells in an in vitro setting.
  • Cell culture medium refers to nutritional compositions used for culturing cells.
  • one or more of the differentiation stages can be performed under feeder-free, serum-free conditions.
  • iPSCs can be seeded in maintenance medium.
  • the maintenance medium includes mTeSR TM 1 or TeSR TM 1 plus from Stem Cell Technologies (Vancouver, Canada), Stem Cell MyeloCult TM H5100 or StemSpan TM SFEM II from Technologies (Vancouver, Canada), E8 medium from Gibco, StemPro TM -34SFM (1X) from Gibco.
  • One or more culture media of the culture platform provided in the present application is a feeder-free environment.
  • the cell culture medium contains a plethora of supplements, such as serum, extracts, growth factors, hormones, cytokines, and the like.
  • the culture platform comprises one or more stage-specific feeder-free, serum-free media, each of which also includes one or more of the following: appropriate nutrients/extracts cytokines and media supplements.
  • Suitable nutrients/extracts may include, for example, DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12), which is a widely used basal medium for supporting the growth of many different mammalian cells; Neurobasal medium is widely used as a basal medium in various cell cultures in humans; L-glutamin.
  • Other media supplements may include, but are not limited to, N2, B27, ⁇ -Me, antioxidants (eg, ascorbic acid).
  • the induction system of the present application comprises one or more of the following cytokines or small molecule compounds: CHIR99021, SB4, VEGF, Forskolin, Deoxyshikonin, Isoarnebin I, Isoarnebin 4, Resveratrol, VPA, SB431542, E-616452, A83-01, UM729, UM171, SR1, these cytokines or small molecule compounds can be commercially available It can be purchased, for example, from R&D Systems (Minneapolis, Minn.), Selleck Company, etc., or it can be natural/recombinant or chemically synthesized.
  • cytokines or small molecule compounds CHIR99021, SB4, VEGF, Forskolin, Deoxyshikonin, Isoarnebin I, Isoarnebin 4, Resveratrol, VPA, SB431542, E-616452, A83-01, UM729, UM171, SR1, these cytokines or small molecule compounds can be
  • the culture system of the present application comprises one or more small molecule compounds: GSK3 ⁇ inhibitors (such as CHIR99021, SB216763, CHIR-98014, LY2090314, BIO-acetoxime, Alsterpaullone, CP21R7), VEGF pathway activators (such as arnebin, Deoxyshikonin, Isoarnebin I, Isoarnebin 4), cAMP Pathway activators (eg Forskolin), Notch signaling pathway activators (eg Resveratrol, VPA), TGF ⁇ receptor/ALK inhibitors (eg SB431542, E-616452, A83-01), hematopoietic stem cell pro-proliferative molecules (eg UM729, UM171, SR1), HIF1a activators (eg Roxadustat, IOX2, DMOG, PT-2385, Belzutif an, PX-478, LW6, Oltipraz, Daprodustat, BAY87-2243,
  • maintenance refers to the persistence, propagation (growth) and/or differentiation of cells outside a tissue or organism, eg, in sterile plastic (or coated plastic) cell culture dishes or flasks. "Cultivating” or “maintaining” may utilize a culture medium as a source of nutrients, hormones, and/or other factors that assist in the propagation and/or persistence of cells.
  • the iHSC-A medium When the iHSC-A medium is used in each culture method and culture platform of the present application, the iHSC-A contains SB4 and CHIR99021.
  • the concentration of Compound A in each medium used is in the range of 0.1-20 ⁇ M.
  • the concentration range of Compound A in the medium is 0.1-20 ⁇ M, preferably, 0.1-15 ⁇ M, more preferably, 0.1-10 ⁇ M.
  • the concentration range of SB4 in each medium used is 0.1-20 ⁇ M.
  • the concentration range of SB4 in the medium is 0.1-20 ⁇ M, preferably 0.1-15 ⁇ M, more preferably 0.1-10 ⁇ M, such as 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2 .4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.2 ⁇ M, 4.3 ⁇ M, 4.4 ⁇
  • the concentration range of CHIR99021 in each medium used is 0.1-20 ⁇ M.
  • the concentration range of SB4 in the medium is 0.1-20 ⁇ M, preferably 0.1-15 ⁇ M, more preferably 0.1-10 ⁇ M, such as 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M,
  • the degree of differentiation of the cell population at each step can be determined by monitoring and/or detecting the expression of one or more cell markers in the differentiated cell population. For example, an increase in expression of a marker characteristic of a more differentiated cell type or a decrease in expression of a marker characteristic of a less differentiated cell type can be determined.
  • Expression of cellular markers can be determined by any suitable technique, including: immunocytochemistry, immunofluorescence, RT-PCR, western blotting, fluorescence activated cell sorting (FACS), and enzymatic analysis.
  • FACS fluorescence activated cell sorting
  • enzymatic analysis for example, a cell described herein that does not express a marker may exhibit active transcription and intracellular expression of the marker gene, but detectable levels of the marker may not be present on the surface of the cell.
  • Partially differentiated cell populations generated by the steps in the methods described herein e.g. non-functional T cells, such as iPSCs, mesoderm, hematopoietic endothelial, HSC, MPP, HPC, T progenitor cells
  • non-functional T cells such as iPSCs, mesoderm, hematopoietic endothelial, HSC, MPP, HPC, T progenitor cells
  • Partially differentiated cells can be cultured, maintained or expanded prior to the next differentiation step.
  • Partially differentiated cells can be expanded by any convenient technique.
  • the partially differentiated cell population may comprise 1% or more, 5% or more, 10% or more, 15% or more partially differentiated cells after culturing in the medium. Portions of differentiated cell populations can be purified, if desired, by any convenient technique such as MACs or FACS.
  • the present application provides methods and compositions for differentiating and expanding hematopoietic cell lineages from pluripotent stem cells in monolayer culture, comprising contacting pluripotent stem cells with an inducing composition I comprising small molecules SB4 and CHIR99021, obtaining and expanding hematopoietic-related mesoderm cells without forming embryoid bodies.
  • the present application provides methods and compositions for differentiating and expanding hematopoietic cell lineages from pluripotent stem cells in monolayer culture, which comprises contacting pluripotent stem cell-derived mesoderm cells with an induction composition II comprising small molecules Deoxyshikonin, Isoarnebin I and/or Isoarnebin 4, and Forskolin, thereby obtaining and expanding hematopoietic endothelium without forming embryoid bodies.
  • an induction composition II comprising small molecules Deoxyshikonin, Isoarnebin I and/or Isoarnebin 4, and Forskolin
  • the present application provides methods for obtaining from Methods and compositions for pluripotent stem cell differentiation and expansion of hematopoietic lineages, comprising contacting pluripotent stem cell-derived hematopoietic endothelium with an inducing composition III comprising small molecules Resveratrol and SB431542, optionally UM729, to obtain and expand hematopoietic stem cells without forming embryoid bodies.
  • the present application provides methods and compositions for differentiating and expanding hematopoietic cell lineages from pluripotent stem cells in monolayer culture, comprising contacting hematopoietic-related mesoderm cells derived from pluripotent stem cells with an inducing composition V comprising small molecules Resveratrol and SB431542, optionally Forskolin, optionally Deoxyshikonin, Isoarnebin I and/or Isoarnebin 4, and obtaining and expanding hematopoietic stem cells without forming embryoid bodies .
  • the iHSC-B comprises VEGF and Deoxyshikonin.
  • the iHSC-B comprises VEGF and Isoarnebin I.
  • the iHSC-B comprises VEGF, Deoxyshikonin and Forskolin.
  • the iHSC-B comprises Deoxyshikonin and Forskolin.
  • the iHSC-B comprises VEGF and Forskolin.
  • the iHSC-B medium when used in each culture method and culture platform of the present application, in a specific embodiment, the iHSC-B comprises VEGF, Isoarnebin I and Forskolin.
  • the iHSC-B medium when used in each culture method and culture platform of the present application, in a specific embodiment, the iHSC-B comprises Isoarnebin I and Forskolin.
  • the iHSC-B medium when used in each culture method and culture platform of the present application, in a specific embodiment, the iHSC-B comprises Isoarnebin 4 and Forskolin.
  • the iHSC-C medium when used in each culture method and culture platform of the present application, in a specific embodiment, the iHSC-C comprises Resveratrol and SB431542.
  • the iHSC-C medium when used in each culture method and culture platform of the present application, in a specific embodiment, the iHSC-C comprises Resveratrol, SB431542 and UM729.
  • the iHSC-D medium comprises Forskolin, Resveratrol, SB431542, VEGF and Deoxyshikonin.
  • the iHSC-D medium comprises Forskolin, Resveratrol, SB431542, and Deoxyshikonin.
  • the iHSC-D medium comprises Forskolin, Resveratrol, SB431542, and VEGF.
  • the iHSC-D medium comprises Forskolin, Resveratrol, SB431542, and Isoarnebin I.
  • the iHSC-D medium comprises Forskolin, Resveratrol, SB431542, and Isoarnebin 4.
  • the iHSC-D medium comprises Forskolin, Resveratrol, SB431542, VEGF and Isoarnebin I.
  • iHSC-B or iHSC-D comprising 1-10 ⁇ M Deoxyshikonin; preferably, 1-6 ⁇ M Deoxyshikonin, more preferably, 2-6 ⁇ M Deoxyshikonin, or comprising 1-20 ⁇ M Isoarnebin I, preferably, 5-15 ⁇ M Isoarnebin I, more preferably, 8-15 ⁇ M Isoarnebin I, or comprising 1 -20 ⁇ M Isoarnebin 4, preferably, 5-15 ⁇ M Isoarnebin 4, more preferably, 8-15 ⁇ M Isoarnebin 4; also include 1-20 ⁇ M Forskolin; preferably, 2-10 ⁇ M Forskolin more preferably, 2-4 ⁇ M Forskolin; also include StemPro34.
  • the concentration range of Deoxyshikonin in each medium used is 1-20 ⁇ M.
  • the concentration range of Deoxyshikonin in the medium is 2-15 ⁇ M, preferably, 3-18 ⁇ M, for example, it can be 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2 .4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.2 ⁇ M, 4.3 ⁇ M, 4.4 ⁇ M M, 4.5
  • the concentration range of Isoarnebin I in each medium used is 1-20 ⁇ M.
  • the range of Isoarnebin concentration in the medium is 2-15 ⁇ M, preferably, 3-18 ⁇ M, for example, it can be 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.2 ⁇ M, 4.3 ⁇ M, 4.4 ⁇ M,
  • the concentration range of Isoarnebin 4 in each medium used is 1-20 ⁇ M.
  • the range of Isoarnebin concentration in the medium is 2-15 ⁇ M, preferably, 3-18 ⁇ M, for example, it can be 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.2 ⁇ M, 4.3 ⁇ M, 4.4 ⁇ M,
  • the concentration range of Forskolin in each medium used is 0.2-10 ⁇ M.
  • the concentration range of Forskolin in the medium is 0.2-10 ⁇ M, preferably, 0.5-8 ⁇ M, more preferably, 1-5 ⁇ M, such as 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2 .3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M,
  • the concentration range of Resveratrol in each medium used is 2-20 ⁇ M.
  • the concentration range of Resveratrol in the medium is 3-18 ⁇ M, preferably, 5-15 ⁇ M, such as 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.2 ⁇ M, 4.3 ⁇ M, 4.4 ⁇ M, 4.5 ⁇ M, 4.6 ⁇ M, 4.7 ⁇ M, 4.8 ⁇ M, 4.9 ⁇ M, 5.0 ⁇ M, 5.1 ⁇ M, 5.2 ⁇ M, 5.3 ⁇ M, 5.4 ⁇ M, 5.5 ⁇ M, 5.6 ⁇ M,
  • the concentration range of SB431542 in each medium used is 2-20 ⁇ M.
  • the concentration range of SB431542 in the culture medium is 3-18 ⁇ M, preferably 5-15 ⁇ M, for example, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M, 3.6 ⁇ M, 3.7 ⁇ M, 3.8 ⁇ M, 3.9 ⁇ M, 4.0 ⁇ M, 4.1 ⁇ M, 4.2 ⁇ M, 4.3 ⁇ M, 4.4 ⁇ M, 4.5 ⁇ M, 4.6 ⁇ M, 4.7 ⁇ M, 4.8 ⁇ M, 4.9 ⁇ M, 5.0 ⁇ M, 5.1 ⁇ M, 5.2 ⁇ M, 5.3 ⁇ M, 5.4 ⁇ M, 5.5 ⁇ M, 5.6 ⁇ M
  • the concentration range of UM729 in each medium used is 0.1-5 ⁇ M.
  • the concentration range of UM729 in the culture medium is 0.3-4 ⁇ M, preferably, 0.5-4.5 ⁇ M, such as 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1 .5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.1 ⁇ M, 2.2 ⁇ M, 2.3 ⁇ M, 2.4 ⁇ M, 2.5 ⁇ M, 2.6 ⁇ M, 2.7 ⁇ M, 2.8 ⁇ M, 2.9 ⁇ M, 3.0 ⁇ M, 3.1 ⁇ M, 3.2 ⁇ M, 3.3 ⁇ M, 3.4 ⁇ M, 3.5 ⁇ M M, 3.6
  • the monolayer culture platform facilitates the acquisition of hematopoietic-associated mesoderm, hematopoietic endothelium, and hematopoietic stem cells, leading to the derivation of hematopoietic stem cells and differentiated progeny such as T, B, NKT, and NK cells.
  • the demonstrated monolayer differentiation strategy combines enhanced differentiation efficiency with massive expansion to deliver therapeutically relevant quantities of pluripotent stem cell-derived hematopoietic cell lineages for a variety of therapeutic applications.
  • the present application discloses that monolayer cultures using the methods provided herein generate hematopoietic cell lineages capable of a full range of in vitro differentiation, ex vivo regulation, and long-term hematopoietic self-renewal, reconstitution, and engraftment in vivo.
  • the inducing composition comprising one or more selected from small molecules provided by the present application is a safe, efficient and low-cost hematopoietic cell lineage differentiation method.
  • the present application relates to a multi-stage process of differentiation of naive pluripotent stem cells to non-pluripotent or partially differentiated cells, comprising, Hematopoietic-related mesoderm cells, hematopoietic endothelium, hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic multipotent progenitor cells (MPP), T progenitor cells, NK progenitor cells; or fully differentiated terminal hematopoietic cells, such as, for example, T cells, B cells, NKT cells or NK cells.
  • the present application also relates to compositions used in the disclosed methods; and cell populations, cell lines or clones produced by the disclosed methods.
  • iPSC-derived hematopoietic lineages were obtained by seeding clonal iPSC cells in media containing a GSK3 ⁇ inhibitor and compound SB4, and using a step-wise strategy to apply a combination of small molecule inducers at early and middle stages of differentiation.
  • the present application enables direct transfer of expanded clonal iPSCs into adherent cultures in a monolayer format for immediate differentiation without the need for EB formation from iPSCs.
  • an inducing composition comprising small molecules SB4 and CHIR99021 can induce differentiation of pluripotent stem cells (including hiPSCs) into hematopoietic-related mesoderm cells under serum-free/feeder-free conditions and in a scalable and monolayer culture platform without the need for EB formation.
  • the inducing composition described herein can also induce differentiation of pluripotent stem cells (including hiPSCs) into mesoderm cells in the context of EB formation.
  • the mesoderm is a stable and efficient mesoderm cell that is conducive to differentiation of hematopoietic cell lineage.
  • methods and compositions for further inducing differentiation of the mesoderm cells to generate hematopoietic endothelium, hematopoietic stem cells, and MPP to generate hematopoietic cell lineages are included.
  • one or more stages of iPSC differentiation described above can be performed under feeder-free conditions.
  • feeder-free conditions can be in forms including, but not limited to, monolayer culture and suspension culture.
  • the differentiation of the pluripotent stem cells to mesoderm cells is performed under monolayer feeder-free conditions.
  • differentiation of mesodermal cells to hematopoietic endothelium is performed under monolayer trophoblast-free conditions.
  • differentiation of hematopoietic endothelium to hematopoietic stem cells is performed under monolayer trophoblast-free conditions.
  • differentiation of hematopoietic stem cells into T progenitor cells or NK progenitor cells is performed under suspension feeder-free conditions, or first performed under monolayer feeder-free conditions, followed by suspension feeder-free conditions. In some embodiments, the differentiation of T progenitor cells to fully differentiated T cells or NK progenitor cells to fully differentiated NK cells is performed under suspension feeder-free conditions, or first performed under monolayer feeder-free conditions followed by suspension feeder-free conditions.
  • compositions and cell culture media contemplated herein can vary and can be optimized for specific culture conditions, including the specific molecules and combinations used, the type of cells cultured in the media, and the specific application.
  • the small molecule is present in the composition at a concentration sufficient to induce hematopoietic endothelium and further hematopoietic differentiation, ultimately achieving efficient and stable hematopoietic cell lineage differentiation.
  • the present application provides a culture platform using feeder-free, serum-free conditions, cytokine-free media that supports direct differentiation of iPSCs in monolayer culture without the need for EBs or aggregation intermediates from iPSCs.
  • pluripotent stem cells primarily rely on trophoblast cells or media preconditioned with trophoblast cells and containing fetal bovine serum; however, this environment may not be suitable for the production of cells for clinical and therapeutic use.
  • the feeder-free environment considered here facilitates the generation of clinical-grade cell lines, particularly hESC, hiPSC, and pluripotent stem cell-derived HSC, MPP, T, B, NKT, or NK cell lines.
  • the trophoblast-free environment is substantially free of human trophoblast cells and is not preconditioned by trophoblast cells Cells, including but not limited to mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells.
  • the trophoblast-free medium is suitable for culturing pluripotent cells, reprogramming cells, single cell culture, dissociation and passage of pluripotent cells, sorting of pluripotent cells, generation of ground state pluripotent cells, maintenance of ground state pluripotency and/or induction of pluripotent cell differentiation.
  • a feeder-free environment is used to induce pluripotency, improve the efficiency of reprogramming, increase or maintain the potency of cells, and/or induce differentiation.
  • trophoblast-free conditions are devoid of cytokines and growth factors, including bFGF.
  • one or more stages of iPSC differentiation described above can be performed under feeder-free conditions.
  • feed-free conditions include, but are not limited to, monolayer and suspension culture formats.
  • differentiation of pluripotent stem cells to hematopoietic-related mesoderm cells is performed under monolayer-free trophoblast conditions.
  • differentiation of mesodermal cells to hematopoietic endothelium is performed under monolayer trophoblast-free conditions.
  • differentiation of hematopoietic endothelium to hematopoietic stem cells, MPPs is performed under monolayer trophoblast-free conditions.
  • differentiation of hematopoietic stem cells into MPP, hematopoietic progenitor cells, T progenitor cells, or NK progenitor cells is performed under suspension feeder-free conditions, or first performed under monolayer feeder-free conditions followed by suspension feeder-free conditions.
  • the differentiation of T progenitor cells to fully differentiated T cells or NK progenitor cells to fully differentiated NK cells is performed under suspension feeder-free conditions, or first performed under monolayer feeder-free conditions followed by suspension feeder-free conditions.
  • Any suitable culture flask or cell culture vessel can be used as a carrier for the cell culture supplemented with minimal medium and/or additional medium.
  • coating the surface of a culture vessel with an adhesion-promoting matrix/substrate e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.
  • an adhesion-promoting matrix/substrate e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.
  • an adhesion-promoting matrix/substrate e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.
  • Suitable substrates for culturing and passaging cells include, but are not limited to, Vitronectin (VTN), Gelatin, Laminin (LN), Fibronectin (FN), Collagen IV (Collagen IV), Elastin, Osteopontin, Thrombospondin, mixtures of matrices produced by naturally occurring cell lines such as MatrigelTM, and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminated single layer.
  • providing feeder-free conditions includes culturing the cells on the matrix-coated surface.
  • culture platforms contemplated herein include a matrix/substrate comprising matrigel, VTN-N, laminin-511 and/or laminin-521.
  • one or more of the differentiation stages described above can be performed under serum-free conditions.
  • serum-free media suitable for cell attachment and/or induction include mTeSR TM 1, mTeSR TM 2, mTeSR plus medium, TeSR TM -AOF medium, StemSpan TM SFEM II, StemSpan CD34+Expansion Supplement (10 x), StemSpan TM -AOF, Primate ES/iPS Cell Medium from ReproCELL (Boston, MA), StemPro TM -34SFM (1X) from Invitrogen (Carlsbad, CA), Essential 8 TM Medium, CST Essential 8 TM Medium, and StemFit basic03 from Ajinomoto.
  • the one or more media of the culture platform is a feeder-free environment, and is optionally substantially free of cytokines and/or growth factors.
  • the cell culture medium contains supplements such as serum, extracts, growth factors, hormones, cytokines, and the like.
  • culture platforms contain one or more stage-specific feeder-free, serum-free media, each of which also contains one or more of the following: nutrients/extracts, growth factors, Hormones, cytokines and media supplements.
  • Suitable nutrients/extracts may include, for example, DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12), which is a widely used basal medium for supporting the growth of many different mammalian cells; KOSR (Knockout Serum Replacement); L-glut; NEAA (Nonessential Amino Acids).
  • Other media supplements may include, but are not limited to, MTG, ITS, ⁇ ME, antioxidants (eg, ascorbic acid).
  • the medium of the present application comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF- ⁇ ), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF) transferrin, various interleukins (such as IL-1 to IL-18), various colony-stimulating factors (such as granulocyte/macrophage colony-stimulating factor (GM-CSF)), various interferons (such as IFN- ⁇ ) and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and
  • EGF
  • the culture medium of the present application comprises one or more bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factor (such as SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (Flt3L); 6.
  • BMP4 bone morphogenetic protein
  • IGF-1 insulin-like growth factor-1
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • hematopoietic growth factor such as SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO
  • Fms-related tyrosine kinase 3 ligand Fms-related tyrosine kinase 3 ligand
  • contacting includes culturing cells in the presence of one or more factors (eg, small molecules, proteins, peptides, etc.).
  • factors eg, small molecules, proteins, peptides, etc.
  • cells are contacted with one or more agents to induce cell differentiation.
  • Such contacting can be performed by introducing one or more reagents into the cells during in vitro culture.
  • contacting can be effected by introducing one or more reagents to the cells in the cell culture medium.
  • Cells can be maintained in a medium comprising one or more agents for a time sufficient for the cells to achieve a desired differentiation phenotype.
  • "contacting" occurs when one or more factors are introduced into a cell via a vector.
  • one or more vectors are introduced by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, vector systems with expression cassettes, or mRNA.
  • one or more stage-specific feeder-free, serum-free media of a culture platform as disclosed herein comprise one or more small molecules.
  • the culture platform comprises a cell culture medium comprising a GSK3 ⁇ inhibitor, a Wnt pathway activator, a VEGF pathway activator, a Notch signaling pathway activator, a TGF ⁇ receptor/ALK inhibitor, and does not comprise a MEK inhibitor.
  • the culture platform contemplated herein also offers a number of advantages by utilizing a homogeneous population of industrial or clinical grade pluripotent cells with reduced spontaneous differentiation and/or attainment of basal state pluripotency.
  • homogeneous iPSCs are maintained in maintenance medium comprising a ROCK inhibitor.
  • the term "homogeneous” refers to a population of cells in which each cell is other cells in the body are identical or substantially identical.
  • a cell is identical to other cells in a population if each cell expresses one or more of the same pluripotency markers contemplated herein, eg, SSEA4, TRA-1-81, TRA-1-60.
  • a population is homogeneous if at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells are identical or substantially identical to other cells in the population.
  • the cell culture medium for the culture platform for generating hematopoietic cell lineages from the hematopoietic endothelium of the present application comprises Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, free or substantially free of GSK3 ⁇ inhibitors, Wnt pathway activators, MEK inhibitors and ROCK inhibitors, cAMP pathway activators and/or VEGF pathway activators.
  • the cell culture medium for the culture platform for generating hematopoietic cell lineages from mesoderm cells of the present application comprises cAMP pathway activators, VEGF pathway activators, Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, free or substantially free of GSK3 ⁇ inhibitors, Wnt pathway activators, MEK inhibitors and ROCK inhibitors.
  • the cell culture medium of the culture platform for generating hematopoietic cell lineage from iPSC cells of the present application comprises compound A, a GSK3 ⁇ inhibitor, a Wnt pathway activator, a cAMP pathway activator, a VEGF pathway activator, a Notch signaling pathway activator and a TGF ⁇ receptor/ALK inhibitor, without or substantially without a MEK inhibitor.
  • the culture platform includes a maintenance medium, a seeding medium for maintaining naive hiPSCs, which medium comprises a ROCH inhibitor.
  • the differentiation inducer composed of small molecule SB4 and CHIR99021 can achieve efficient hematopoietic differentiation in the absence of BMP4 and Activin A, and is hardly affected by cell plating density.
  • VEGF vascular endothelium
  • VEGF pathway activators especially when combined with cAMP activators
  • the term “long-term” is measured based on, but not limited to, the number of passages, generally meaning at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more passages.
  • passaging refers to the act of dividing and spreading cells onto multi-cell culture surfaces or culture flasks when they have proliferated to a desired extent.
  • homogenized iPSCs are maintained in maintenance medium comprising a ROCK inhibitor.
  • R1 is selected from:
  • X1 is selected from H, methyl, ethyl, hydroxy, methoxy, ethoxy, halogen
  • X2 is selected from H, methyl, ethyl, hydroxy, methoxy, ethoxy, halogen
  • X3 is selected from H, methyl, ethyl, hydroxy, methoxy, ethoxy, halogen; Among them, R2 is:
  • the X4, X5, X6, or X7 are selected from H, methyl, ethyl, hydroxyl, methoxy, ethoxy, or halogen, and X4, X5, X6, or X7 can be the same or different;
  • the R2 is selected from any of the following structures:
  • Wnt pathway activator in the present application refers to an activator of the Wnt signaling pathway, including but not limited to Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt1 4.
  • GSK3 ⁇ inhibitor in this application has the effect of activating Wnt pathway, including but not limited to polynucleotides, polypeptides and small molecules.
  • GSK3 ⁇ inhibitors can reduce GSK3 ⁇ expression and/or GSK3 ⁇ activity.
  • GSK3 ⁇ inhibitors include, but are not limited to, anti-GSK3 ⁇ antibodies, dominant negative GSK3 ⁇ variants, siRNA or shRNA or miRNA or antisense nucleic acids targeting GSK3 ⁇ .
  • GSK3 ⁇ inhibitors include, but are not limited to: Camparolone, 1-Azacamparolone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, Lithium, SB415286, TDZD-8, BIO, BIO-acetone oxime, (5-methyl- 1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridinecarbazole-cyclopentadienyl ruthenium complex, TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3-,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, ⁇ -4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H
  • the GSK3 ⁇ inhibitor is CHIR99021, BIO, or camparolone. In some examples, the GSK3 ⁇ inhibitor is CHIR99021. In some examples, the GSK3 ⁇ inhibitor is BRD0705. In some examples, the concentration of CHIR99021 is in the range of 0.1-20 ⁇ M, preferably 0.1-15 ⁇ M, more preferably 0.1-10 ⁇ M.
  • VEGF vascular endothelial growth factor
  • VEGF pathway activators include one or more of the following: including but not limited to polynucleotides, polypeptides, and small molecule compounds. Activators of one or more of VEGF121, VEGF145, VEGF162, VEGF165, VEGF183, VEGF189, and VEGF206, including but not limited to.
  • Nucleic acid comprising a nucleotide sequence encoding a VEGF polypeptide, a polypeptide comprising an amino acid sequence of a VEGF polypeptide, a nucleic acid comprising a nucleotide sequence encoding an activated VEGF receptor, a polypeptide comprising an amino acid sequence of an activated VEGF receptor, a small organic molecule that promotes VEGF signaling, a small organic molecule that inhibits the expression or activity of a VEGF antagonist, an antisense oligonucleotide that inhibits the expression of a VEGF antagonist, a ribozyme that inhibits the expression of a VEGF antagonist, an RNAi construct or siRNA or shRNA that inhibits the expression of a VEGF antagonist, binds to and inhibits the expression of a VEGF antagonist Antibody with VEGF antagonist activity, nucleic acid comprising nucleotide sequence encoding VEGF protein polypeptide, polypeptide comprising amino acid
  • Isoarnebin I also known as ⁇ , ⁇ -Dimethylacrylshikonin (Isoarnebin I) is a naphthoquinone derivative extracted from Arnebia nobilis.
  • the structure of Isoarnebin I is as follows:
  • Deoxyshikonin isolated from Lithospermum erythrorhizon, increases the expression of VEGF-C and VEGF-A mRNA in HMVEC-dLy.
  • the structure of Deoxyshikonin is as follows:
  • Isoarnebin 4 also known as Shikonin, is a potent and specific inhibitor of Pyruvate kinase M2 (PKM2).
  • PPM2 Pyruvate kinase M2
  • Activators of the cAMP pathway include one or more of the following: a nucleic acid comprising a nucleotide sequence encoding a cAMP polypeptide, a polypeptide comprising an amino acid sequence of a cAMP polypeptide, a nucleic acid comprising a nucleotide sequence encoding an activated cAMP receptor, a polypeptide comprising an amino acid sequence that activates a cAMP receptor, a small organic molecule that promotes cAMP signaling, a small organic molecule that inhibits the expression or activity of a cAMP antagonist, an antisense oligonucleotide that inhibits the expression of a cAMP antagonist, a ribozyme that inhibits the expression of a cAMP antagonist, an inhibitor of a cAMP antagonist
  • Formkolin also known as Colforsin, is an adenylate cyclase activator with a molecular formula of C22H34O7 and a structure as shown below:
  • PACAP 1-38 which stands for Pituitary Adenylate Cyclase Activating Polypeptide 38, is a highly potent PACAP receptor agonist capable of stimulating adenylate cyclase.
  • the structure of PACAP 1-38 is shown below:
  • Notch includes all members of the Notch receptor family, including but not limited to Notch1.
  • Notch signaling pathway activators include, but are not limited to, agonists of Notch receptors.
  • a Notch signaling pathway agonist will bind to the Notch receptor and also initiate or mediate signaling events associated with the Notch receptor, such as causing the intracellular domain of Notch to be cleaved and translocated to the nucleus.
  • Notch signaling pathway activators include one or more of the following: nucleic acids comprising nucleotide sequences encoding Notch polypeptides, polypeptides comprising amino acid sequences of Notch polypeptides, nucleic acids comprising nucleotide sequences encoding activated Notch receptors, polypeptides comprising amino acid sequences of activated Notch receptors, small organic molecules that promote Notch signal transduction, small organic molecules that inhibit the expression or activity of Notch antagonists, antisense oligonucleotides that inhibit the expression of Notch antagonists, ribozymes that inhibit the expression of Notch antagonists, inhibit Notch antagonism
  • Notch signaling pathway activators include but not limited to Jag1, Jag2, DLL-1, DLL-3 and DLL-4. Notch signaling pathway activators include, but are not limited to, those disclosed in EP 2606884, US 6689744 and US5780300, the disclosures of which are incorporated herein by reference.
  • one or more Notch ligands can be introduced as soluble peptides, or immobilized on a solid material. Solid materials may include, but are not limited to, polystyrene plates or beads. The beads used for Notch ligand immobilization can be agarose beads, magnetic beads and latex beads. In some examples, Notch ligand peptides are conjugated/immobilized to beads.
  • Notch ligand peptides are conjugated/immobilized to the surface of a polystyrene plate. In some examples, the immobilization of the Notch ligand is non-covalent. In some examples, the Notch ligand peptide is presented by the cell. In some examples, the Notch signaling pathway activator includes Resveratrol and/or VPA.
  • Resveratrol also known as SRT501 or trans-Resveratrol, has a broad range of targets including cyclooxygenases, lipoxygenases, sirtuins and other proteins.
  • SRT501 SRT501 or trans-Resveratrol
  • targets including cyclooxygenases, lipoxygenases, sirtuins and other proteins.
  • the structure of Resveratrol is as follows:
  • VPA Valproic acid
  • HDAC histone deacetylase
  • TGF ⁇ receptor/ALK inhibitor ie includes TGF ⁇ receptor inhibitors that act by inhibiting the ALK pathway. Including antibodies against TGF ⁇ receptors (such as ALK5), dominant negative variants of TGF ⁇ receptors, antisense nucleic acids that inhibit the expression of TGF ⁇ receptors, or small molecular compounds that inhibit TGF ⁇ receptor/ALK signaling pathways.
  • TGF ⁇ receptor/ALK inhibitors include, but are not limited to, SB431542; A-83-01, also known as 3-(6-methyl-2-pyridine)-N-phenyl-4-(4-quinoline)-1H-pyrazole-1-thioamide; RepSox (ie E616452); ; Wnt3a/BIO; GW788388 (- ⁇ 4-[3-(pyridin-2-yl)-lH-pyrazol-4-yl]pyridin-2-yl ⁇ -N-(tetrahydro-2H-pyran-4-yl)benzamide); SM16 (see, e.g., Suzuki et al., Cancer Research 67(5):2351-2359 (2007)); IN-1130(3 -((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide); GW6604(2-phenyl-4-(3-pyridin
  • TGF ⁇ receptor/ALK inhibitors include, but are not limited to: TGF ⁇ receptor inhibitors, SMAD 2/3 phosphorylation inhibitors, SMAD 2/3 and SMAD 4 interaction inhibitors, and SMAD 6 and SMAD 7 activators/agonists.
  • TGF receptor inhibitors include, but are not limited to, SU5416; 2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-picoline hydrochloride (SB-505124); lerdelimumb (CAT-152); -12009; AP-11014; LY550410; LY580276; LY364947; LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; .
  • TGF ⁇ receptor/ALK inhibitors include SB431542, A-83-01, E-616452, or combinations thereof.
  • SB431542 which is a TGF ⁇ receptor/ALK5 inhibitor, has the following structure:
  • A-83-01 which is an inhibitor of TGF- ⁇ type I receptor (ALK5-TD), also inhibits the transcription induced by ALK4-TD and ALK7-TD.
  • ALK5-TD TGF- ⁇ type I receptor
  • E-616452 also known as RepSox, SJN 2511, ALK5 Inhibitor II, is a potent and selective TGF ⁇ R-1/ALK5 inhibitor.
  • the structure of E-616452 looks like this:
  • the pro-proliferation molecules of hematopoietic stem cells include SCF, FL, IL-1, IL-12 and other cytokines, or small molecule compounds.
  • the hematopoietic stem cell pro-proliferation molecule comprises UM729, UM171, SR1 or a combination thereof.
  • UM729 a pyrimidine-[4,5-b]-indole derivative, enhances the self-renewal of human hematopoietic stem cells in vitro.
  • the structure of UM729 is as follows:
  • UM171 is a potent agonist of human hematopoietic stem cell self-renewal independent of AhR inhibition.
  • SR1 StemRegenin 1.
  • the structure of SR1 is as follows:
  • the present application provides that the sorted or enriched pluripotent stem cell-derived HSC and MPP cells are further differentiated into various specific cell types along the hematopoietic lineage, including T cells and NK cells.
  • T cells after enrichment, iHSC and MPP cells were transferred to suspension culture without trophoblast cells or adherent culture with OP9 stromal cells or matrigel-coated surface. Cultures were supplemented with iTC-A containing soluble DLL1 and DLL4. After about 10 days, the culture environment was replaced with iTC-B to complete T cell maturation. Approximately 30-40 days (after the original induction of differentiation), T cell composition was assessed, including surface expression of CD3, CD7, TCR ⁇ , CD4, and CD8.
  • the resulting CD34 positive cells confer in vitro differentiation capacity of distinct T cell populations defined by the expression of CD4 and CD8 from the CD7 population.
  • HSC cells were treated with differentiation medium including IL15, iNK-A medium for approximately 10 days, and switched to iNK-B medium for an additional 10-20 days. Cultivation is carried out in suspension.
  • the multi-stage culture platform described here demonstrates the use of sequential differentiation methods to derive hematopoietic stem cells from a variety of stem cells.
  • the resulting CD34 positive hematopoietic stem cells can be maintained in suspension culture for scaling and give rise to a variety of hematopoietic cell lineages including hematopoietic stem cells, MPPs, hematopoietic progenitors, T cells and NK cells.
  • the resulting CD34-positive permanent hematopoietic stem cells were shown to respond to pharmacological modulation by upregulating the immunomodulatory surface protein PDL1.
  • the resulting CD34 positive cells are capable of reconstituting both myeloid and lymphoid populations in vivo. Permanent hematopoietic stem cells from various populations, including pluripotent stem cells, are ideal candidates for patient-specific therapeutic and regenerative medicine applications.
  • the present application provides a medium iHSC-A for obtaining mesoderm cells from pluripotent stem cells including iPSCs.
  • the iPSCs are naive iPSCs.
  • the culture medium comprises a GSK3 ⁇ inhibitor and the small molecule SB4.
  • the medium contains GSK3 ⁇ inhibitor and small molecule SB4 and iHSC-A basic medium, and the medium does not contain or substantially does not contain BMP4, bFGF, LIF, Activin A.
  • the GSK3 ⁇ inhibitor in the culture medium activates the Wnt signaling pathway.
  • the GSK3 ⁇ inhibitor is CHIR99021, BIO, or camparolone.
  • the GSK3 ⁇ inhibitor is CHIR99021.
  • the medium iHSC-A comprises extracellular matrix proteins.
  • the extracellular matrix protein includes matrigel, VTN-N, Laminin-511, Laminin-521, or combinations thereof.
  • the culture medium iHSC-A herein contains basic medium, additional medium, small molecules and/or cytokines in the concentration ranges shown in Table 1.
  • TRA-1-85 is stably and highly expressed during directed mesoderm differentiation.
  • membrane expression of SSEA4 is progressively reduced during committed mesoderm differentiation. The gradual decrease of SSEA4 indicates that induced pluripotent stem cells are gradually differentiating and losing pluripotency.
  • the expression of CD56 exhibits an initial baseline expression level during the directed differentiation of mesoderm, then decreases and returns to a state of even a high expression level. This shows that in the process of directed hematopoietic-related mesoderm differentiation, the expression of CD56 is generally low in the state of stem cells. As the differentiation progresses, CD56 presents a dynamic change process of decreasing first and then increasing, and showing a high expression level when reaching the mesoderm. The mesoderm induced and differentiated by medium iHSC-A can be effectively differentiated into hematopoietic endothelium through various methods.
  • the present application provides a medium iHSC-B for obtaining hematopoietic endothelium from mesoderm cells.
  • the culture medium contains high concentrations of VEGF and/or VEGF pathway activators, and optionally a cAMP pathway activator.
  • the culture medium comprises a high concentration of VEGF and/or a VEGF pathway activator, a cAMP pathway activator, and iHSC-B minimal medium, and the medium is free or substantially free of GSK3 ⁇ inhibitors, Activin A, and bFGF.
  • the iHSC-B minimal medium is StemPro34 medium.
  • the medium contains 30-200 ng/ml VEGF, preferably 50-200 ng/ml VEGF, more preferably 100-200 ng/ml VEGF.
  • the VEGF pathway activator is Deoxyshikonin.
  • the VEGF pathway activator is Isoarnebin I.
  • the VEGF pathway activator is Isoarnebin 4.
  • the cAMP pathway activator is Forskolin.
  • the cAMP pathway activator is PACAP 1-38.
  • the iHSC-B comprises Forskolin and VEGF.
  • the iHSC-B comprises Forskolin and Deoxyshikonin.
  • iHSC-B comprises Forsklin and Isoarnebin4. In some examples, iHSC-B comprises Forskolin and Isoarnebin I.
  • the medium iHSC-B comprises extracellular matrix proteins. In some examples, the extracellular matrix protein includes matrigel, VTN-N, Laminin-511, Laminin-521, or combinations thereof. In the specific example provided in this application, the culture medium iHSC-B herein contains basic medium, additional medium, small molecules and/or cytokines in the concentration ranges shown in Table 2.
  • Hematopoietic-related mesoderm cells derived from various induction differentiation methods can be cultured by iHSC-B Support the hematopoietic endothelium.
  • the hematopoietic endothelium induced and differentiated by the medium iHSC-B can be effectively differentiated into hematopoietic stem cells through various methods.
  • the present application provides a medium iHSC-C for obtaining hematopoietic stem cells from hematopoietic endothelium.
  • the medium iHSC-C comprises a Notch signaling pathway activator and a TGF ⁇ receptor/ALK inhibitor, and optionally a hematopoietic stem cell pro-proliferative molecule.
  • the culture medium iHSC-C comprises Notch signaling pathway activator, TGF ⁇ receptor/ALK inhibitor and iHSC-C minimal medium, and the medium optionally comprises hematopoietic stem cell proliferation-promoting molecules, but does not contain or substantially does not contain GSK3 ⁇ inhibitor, VEGF pathway activator, cAMP pathway activator and/or BMP activator.
  • the iHSC-C minimal medium is StemPro34 medium.
  • the Notch signaling pathway activator is Resveratrol.
  • the Notch signaling pathway activator is VPA.
  • the TGF ⁇ receptor/ALK inhibitor is SB431542.
  • the TGF ⁇ receptor/ALK inhibitor is E-616452.
  • the TGF ⁇ receptor/ALK inhibitor is A83-01.
  • the hematopoietic stem cell pro-proliferative molecule is UM729.
  • the hematopoietic stem cell pro-proliferative molecule is UM171.
  • the hematopoietic stem cell pro-proliferative molecule is SR1.
  • the iHSC-C comprises Resveratrol, SB431542, and UM729.
  • the medium iHSC-C comprises extracellular matrix proteins.
  • the extracellular matrix protein includes matrigel, VTN-N, Laminin-511, Laminin-521, or combinations thereof.
  • the medium iHSC-C herein contains the basic medium, additional medium, small molecules and/or cytokines in the concentration ranges shown in Table 3.
  • Hematopoietic endothelial cells derived from a variety of induction differentiation methods can be cultured with iHSC-C to obtain hematopoietic stem cells.
  • the hematopoietic stem cells induced and differentiated by the medium iHSC-C can be effectively differentiated into T cells and NK cells through various methods.
  • the present application provides a medium iHSC-D for obtaining hematopoietic stem cells from hematopoietic-related mesoderm cells.
  • the medium iHSC-D contains high concentrations of VEGF and/or VEGF pathway activators, Notch signaling pathway activators, TGF ⁇ receptor/ALK inhibitors and cAMP pathway activators.
  • the culture medium iHSC-D comprises a high concentration of VEGF and/or VEGF pathway activator, Notch signaling pathway activator, TGF ⁇ receptor/ALK inhibitor and cAMP pathway activator and iHSC-D basic medium, and the medium optionally comprises hematopoietic stem cell proliferation-promoting molecules, but does not contain or substantially does not contain bFGF, GSK3 ⁇ inhibitor and/or BMP activator.
  • the medium iHSC-D comprises 30-200 ng/ml VEGF, preferably, 50-200 ng/ml VEGF, more preferably, 100-200 ng/ml VEGF.
  • the VEGF pathway activator is Deoxyshikonin.
  • the VEGF pathway activator is Isoarnebin I. In some examples, the VEGF pathway activator is Isoarnebin 4. In some examples, the cAMP pathway activator is Forskolin. In some examples, the cAMP pathway activator is PACAP 1-38. In some examples, the Notch signaling pathway activator is Resveratrol. In some examples, the Notch signaling pathway activator is VPA. In some examples, the TGF ⁇ receptor/ALK inhibitor is SB431542. In some examples, the TGF ⁇ receptor/ALK inhibitor is E-616452. In some examples, the TGF ⁇ receptor/ALK inhibitor is A83-01. In some examples, the hematopoietic stem cell pro-proliferative molecule is UM729.
  • the hematopoietic stem cell pro-proliferative molecule is UM171. In some examples, the hematopoietic stem cell pro-proliferative molecule is SR1. In some examples, iHSC-D(VEGF) comprises Forskolin, Resveratrol, SB431542 and VEGF. In some examples, iHSC-D (Deoxyshikonin) comprises Forskolin, Resveratrol, SB431542, and Deoxyshikonin. In some examples, iHSC-D (Isoarnebin I) comprises Forskolin, Resveratrol, SB431542, and Isoarnebin I.
  • iHSC-D(Isoarnebin4) comprises Forskolin, Resveratrol, SB431542 and Isoarnebin4.
  • the iHSC-D minimal medium is StemPro34 medium.
  • the iHSC-D minimal medium is MCH5100 medium.
  • the iHSC-D minimal medium is Stemspan II medium.
  • the medium iHSC-D comprises extracellular matrix proteins.
  • the extracellular matrix protein includes matrigel, VTN-N, Laminin-511, Laminin-521, or combinations thereof.
  • the culture medium iHSC-D herein comprises minimal medium, additional medium, small molecules and/or cytokines in the concentration ranges shown in Table 4.
  • Hematopoietic-related mesoderm cells derived from a variety of induced differentiation methods can be cultured with iHSC-D to obtain hematopoietic stem cells.
  • Hematopoietic stem cells induced by medium iHSC-D can be effectively differentiated into T cells and NK cells through various methods.
  • a culture platform for obtaining T cells comprising one or more (i) medium, It comprises one or more growth factors and cytokines selected from SCF, Flt3L, IL7 and IGF and iTC minimal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators, wherein the medium does not contain BMP activators and is suitable for generating T cells from T progenitor cells; (ii) a medium comprising BMP activators, one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and iTC minimal medium, wherein Suitable for generating T progenitor cells from iHSCs.
  • medium comprises one or more growth factors and cytokines selected from SCF, Flt3L, IL7 and IGF and iTC minimal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators,
  • Another aspect of the present application provides a culture platform for obtaining T cells comprising one or more (i) medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7 and IGF and iTC minimal medium comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators, wherein the medium does not contain BMP activators and is suitable for generating T cells from T progenitor cells; (ii) medium comprising BMP activators one or more selected from SC F, Growth factors and cytokines of Flt3L and IL7 and iTC minimal medium, where the medium is suitable for generating T progenitor cells from iHSCs.
  • medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7 and IGF and iTC minimal medium comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators,
  • One aspect of the present application provides a culture platform for obtaining T progenitor cells, comprising one or more (i) medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and iTC minimal medium, wherein the medium is suitable for producing T progenitor cells from iHSC.
  • the culture platform for obtaining NK cells comprises (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF and IL7 and iNK minimal medium comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators, wherein the medium is free of BMP activators and is suitable for generating NK cells from NK progenitor cells.
  • the culture platform comprising medium (i) further comprises (ii) medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and iNK minimal medium, wherein medium (ii) is suitable for producing NK progenitor cells from iHSCs.
  • a culture platform for obtaining iHSCs which comprises one or more (i) medium comprising one or more cytokines selected from SCF, GM-CSF, IL3, IL6, FLT-3L and TPO and iHSC-C minimal medium, the latter comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, and the medium optionally comprises hematopoietic stem cell pro-proliferation molecules, but does not contain or substantially does not contain GSK3 ⁇ inhibitors, VEGF pathway activators, cAMP pathway activation (ii) a culture medium comprising a high concentration of VEGF and/or a VEGF pathway activator, a cAMP pathway activator, and iHSC-B minimal medium without or substantially free of GSK3 ⁇ inhibitors, Activin A and/or bFGF, said medium being suitable for producing hematopoietic endothelium from mesoderm cells; and (iii) a medium comprising a cytokines selected from S
  • the iPSCs are naive iPSCs.
  • the initial iPSC plating density is about 2.5x10 4 cells/cm 2 .
  • the initial iPSC plating density is about 10x10 4 cells/cm 2 .
  • hematopoietic stem cells can emerge as early as day 8 after hematopoietic differentiation. In some embodiments, hematopoietic stem cells can appear as early as day 8 after hematopoietic differentiation, and the hematopoietic stem cells can be continuously collected until day 20.
  • the amount of induced hematopoietic stem cells can reach 10 6 in each well of a 24-well plate, and can be expanded in multiple generations with a high proliferation rate.
  • the iHSCs prepared by the iHSC platform continue to differentiate into T and NK cells.
  • iHSC-B is When combined with VEGF+Forskolin, the iHSC platform can obtain CD34+iHSC cells with a positive rate of up to 80%.
  • HE obtained from iHSC culture platform 1 differentiation accounted for about 80%
  • arteriovenous endothelium mainly arterial endothelium
  • This greatly facilitates efficient endothelial hematopoietic conversion and lays a solid platform foundation for the subsequent efficient differentiation of induced hematopoietic stem cells.
  • the iHSC platform can stably and efficiently differentiate to obtain CD43+CD45+iHSC.
  • CD38 Since CD38 is negatively expressed in iHSC and early lymphoid, it is positive in early myeloid.
  • iHSC-B are combinations of VEGF+Forskolin, Deoxyshikonin+Forskolin, Isoarnebin I+Forskolin, and Isoarnebin 4+Forskolin, the iHSC platform can stably and efficiently differentiate to obtain CD43+CD45+iHSCs, of which the proportion of CD38+myeloid cells is less than 1%, which shows that this iHSC platform can be used for myeloid differentiation and/or lymphoid differentiation , or preferentially for lymphoid differentiation.
  • the present application provides a culture platform for obtaining iHSCs, which comprises one or more (i) culture medium, which contains high concentration VEGF and/or VEGF pathway activator, Notch signaling pathway activator, TGF ⁇ receptor/ALK inhibitor and cAMP pathway activator and iHSC-D basic medium, and the medium optionally comprises hematopoietic stem cell proliferation-promoting molecules, but does not contain or substantially does not contain bFGF, GSK3 ⁇ inhibitor and/or BMP activator, and the medium is suitable for producing iHSC from mesoderm cells; and (ii) ) medium comprising a GSK3 ⁇ inhibitor and small molecule SB4 and iHSC-A minimal medium, free or substantially free of BMP4, Activin A, bFGF and/or LIF, suitable for generating mesoderm cells from iPSCs.
  • culture medium which contains high concentration VEGF and/or VEGF pathway activator, Notch signaling pathway activator, TGF ⁇ receptor/ALK inhibitor and
  • the iPSCs are naive iPSCs.
  • the initial iPSC plating density is about 2.5x10 4 cells/cm 2 .
  • the initial iPSC plating density is about 10x10 4 cells/cm 2 .
  • hematopoietic stem cells can emerge as early as day 8 after hematopoietic differentiation.
  • hematopoietic stem cells can appear as early as day 9 after hematopoietic differentiation, and the hematopoietic stem cells can be continuously collected until day 20.
  • the amount of induced hematopoietic stem cells can reach 10 6 in each well of a 24-well plate, and can be expanded in multiple generations with a high proliferation rate.
  • the iHSC-D basic medium is that the iHSCs prepared from the MCH5100 iHSC platform are more inclined or more conducive to differentiation into macrophages or monocytes.
  • the iHSC-D basic medium is StemSpan SFEM II or StemPro34 iHSC platform prepared iHSCs are more inclined or more conducive to differentiation into T and NK cells.
  • the iHSC-D minimal medium is used to further differentiate iHSCs prepared from the MCH5100 iHSC platform into macrophages or monocytes.
  • the iHSC-D basic medium is the iHSC platform prepared by StemSpan SFEM II or StemPro34, and the iHSCs continue to differentiate into T and NK cells.
  • the iHSC platform can stably and efficiently differentiate to obtain CD43+CD45+iHSC.
  • CD38 is negatively expressed in iHSC and early lymphoid lineage, it is positively expressed in early myeloid lineage.
  • the iHSC platform can stably and efficiently differentiate into CD43+CD45+iHSCs, among which CD38+ myeloid cells account for about 20%, which shows that the iHSCs obtained from this culture platform have a relatively obvious early myeloid differentiation tendency, so this culture platform can be used for myeloid differentiation and/or lymphoid differentiation, or preferably for myeloid differentiation.
  • the iHSC-D basic medium is StemPro34 and StemSpan SFEM II culture conditions
  • the iHSC platform can stably and efficiently differentiate to obtain CD43+CD45+iHSC, wherein, CD38+
  • the proportion of myeloid type cells is about 0% to 8%, which indicates that the culture platform can be used for myeloid differentiation and/or lymphoid differentiation, or preferentially for lymphoid differentiation.
  • the culture platform for obtaining iHSCs comprises (i) medium comprising one or more cytokines selected from SCF, GM-CSF, IL3, IL6, FLT-3L and TPO and iHSC-C minimal medium, the latter further comprising Notch signaling pathway activators and TGF ⁇ receptor/ALK inhibitors, and the medium optionally comprises hematopoietic stem cell pro-proliferation molecules, but does not contain or substantially does not contain GSK3 ⁇ inhibitors, VEGF pathway activators, cAMP pathway activators and/or BMP activator, the medium is suitable for generating iHSCs from hematopoietic endothelium.
  • the culture platform comprising medium (i) further comprises (ii) a medium comprising a high concentration of VEGF and/or a VEGF pathway activator, a cAMP pathway activator, and iHSC-B minimal medium free or substantially free of a GSK3 ⁇ inhibitor, Activin A and/or bFGF, said medium being suitable for generating hematopoietic endothelium from mesoderm cells.
  • the culture platform comprising medium (i) further comprises (iii) medium comprising high concentrations of VEGF and/or VEGF pathway activators, Notch signaling pathway activators, TGF ⁇ receptor/ALK inhibitors and cAMP pathway activators and iHSC-D minimal medium, and the medium optionally comprises hematopoietic stem cell proliferation-promoting molecules, but does not contain or substantially does not contain bFGF, GSK3 ⁇ inhibitors and/or BMP activators, and the medium is suitable for generating iHSCs from mesoderm cells.
  • the culture platform comprising mediums (i) and (ii) further comprises (iv) a medium comprising a GSK3 ⁇ inhibitor and a small molecule SB4 and iHSC-A basic medium without or substantially without BMP4, Activin A, bFGF and/or LIF, said medium being suitable for producing mesoderm cells from iPSCs.
  • the culture platform comprising mediums (i) and (iii) further comprises (iv) a medium comprising a GSK3 ⁇ inhibitor and a small molecule SB4 and iHSC-A minimal medium, and the medium is free or substantially free of BMP4, Activin A, bFGF and/or LIF, said medium being suitable for producing mesoderm cells from iPSCs.
  • the iPSCs are naive iPSCs.
  • the application provides a culture platform for obtaining hematopoietic endothelium, which comprises one or more (i) medium comprising high concentration VEGF and/or VEGF pathway activator, cAMP pathway activator and iHSC-B basic medium, and the medium does not contain or substantially does not contain GSK3 ⁇ inhibitor, Activin A and/or bFGF, said medium is suitable for producing hematopoietic endothelium from mesoderm cells, and (ii) medium, which comprises GSK3 ⁇ inhibitor and small molecule SB4 and iHSC-A basic medium, and The medium is free or substantially free of BMP4, Activin A, bFGF and/or LIF and is suitable for generating mesoderm cells from iPSCs.
  • medium comprising high concentration VEGF and/or VEGF pathway activator, cAMP pathway activator and iHSC-B basic medium, and the medium does not contain or substantially does not contain GSK3 ⁇ inhibitor, Activin A and/or
  • the culture platform for obtaining hematopoietic endothelium comprises (i) a medium comprising a high concentration of VEGF and/or a VEGF pathway activator, a cAMP pathway activator, and iHSC-B minimal medium free or substantially free of GSK3 ⁇ inhibitors, Activin A and/or bFGF, said medium being suitable for producing hematopoietic endothelium from mesoderm cells.
  • the culture platform comprising medium (i) further comprises (ii) medium comprising a GSK3 ⁇ inhibitor and small molecule SB4 and iHSC-A minimal medium without or substantially without BMP4, Activin A, bFGF and/or LIF, said medium being suitable for producing mesoderm cells from iPSCs.
  • Medium iTC-A comprises a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators selected from Jag1, Jag2, DLL-1, DLL-3, and DLL-4; in some instances, the composition is free of VEGF and/or IL15.
  • Medium iTC-B contains one or more A growth factor and cytokine selected from SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators selected from Jag1, Jag2, DLL-1, DLL-3, and DLL-4; in some instances, the composition does not contain a BMP activator.
  • the medium iNK-A contains a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15.
  • the medium iNK-B contains one or more growth factors selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15.
  • the dosage specifically disclosed in the application is a safe and effective dosage obtained by the applicant through research.
  • Those skilled in the art, such as clinicians can determine the dosage for each cycle according to various actual situations, such as the patient's tumor burden, the patient's own physical condition and other factors.
  • the present application provides methods and compositions that enable direct differentiation of pluripotent stem cell monolayers without generating embryoid bodies from pluripotent stem cells, thereby enabling differentiation and expansion of mesoderm cells, HE and iHSCs, T cells, NK cells, where other hematopoietic cell lineages can be obtained in a scalable, reliable format at very high efficiency levels.
  • Example 1 Generation and maintenance of human induced pluripotent stem cells (hiPSCs)
  • various factor combinations including Oct4/Sox2/Klf4 or Oct4/Sox2/Klf4/mp53DD/EBNA1 or Oct4/Sox2/Klf4/Lin28/L-Myc/mp53DD/EBNA1 are used to induce human non-mobilized peripheral blood CD34+ Somatic cells, including stem cells and peripheral blood endothelial precursor cells, reprogram to a pluripotent state.
  • hiPSCs monoclonals obtained from reprogramming in good condition as the initial cells After 14-16 days after induction, gradually pick hiPSCs monoclonals obtained from reprogramming in good condition as the initial cells, and transplant them into an induced pluripotent stem cell culture system such as matrigel, VTN or LN as the extracellular matrix, and mTeSR1Plus or E8 medium as the maintenance medium for acclimatization and expansion.
  • an induced pluripotent stem cell culture system such as matrigel, VTN or LN as the extracellular matrix
  • mTeSR1Plus or E8 medium as the maintenance medium for acclimatization and expansion.
  • the hiPSCs obtained are naive/starting cells.
  • naive hiPSCs are seeded as a monolayer in maintenance medium And allow to expand until about 25% of the plank area is reached.
  • the maintenance medium was switched to the medium shown in Table 1, iHSC-A started hematopoietic differentiation (denoted as day 0).
  • the culture was then switched to the medium iHSC-B shown in Table 2 on day 3-4 after the initiation of differentiation, and switched to the medium iHSC-C shown in Table 3 on day 5-20 after the initiation of differentiation. Attached cultures remained attached and undisturbed during media changes.
  • iHSC culture platform 1 is a hematopoietic culture platform for monolayer culture without serum and feeder layer ( Figure 1).
  • hematopoietic mesoderm differentiation was monitored by lineage markers CD56 and BRACHYURY; on day 4 (this time point can be extended, up to day 8), the directional induction of hematopoietic endothelium was identified by monitoring the expression of CD31, CD144, CD34, CD184, and CD73.
  • hematopoietic endothelial induction After the hematopoietic endothelial induction is completed, by observing the appearance of cells that become round and detached from the adherent form, to the gradual appearance of clusters or scattered hematopoietic stem-like cell populations in the shape of bright beads, it is basically confirmed from the phenotype that they have differentiated to the stage of hematopoietic stem cells. Detection of hematopoietic differentiation potential (CFU monoclonal colony formation).
  • naive hiPSCs were seeded as a monolayer in maintenance medium and allowed to expand until approximately 25% of the plated area was reached.
  • hematopoietic differentiation was initiated by switching the maintenance medium to the medium iHSC-A shown in Table 1.
  • the culture was then switched to the medium iHSC-D shown in Table 4 from day 3 after the initiation of differentiation. Attached cultures remained attached and undisturbed during media changes.
  • iHSC culture platform 2 is a serum-free and feeder-free monolayer culture platform for hematopoietic culture ( Figure 2).
  • hematopoietic mesoderm differentiation was monitored by the lineage markers CD56 and BRACHYURY; on day 4 to day 5 (this time period can be adjusted within 1 day to 4 days), the directional induction of hematopoietic endothelium was identified by monitoring the expression of CD31, CD144, CD34, CD184, and CD73.
  • hematopoietic endothelial induction After the hematopoietic endothelial induction is completed, by observing the appearance of cells that are rounded and detached from the stickers, to the gradual appearance of hematopoietic stem-like cell populations in the shape of bright beads or clusters or scattered, it can basically be confirmed from the phenotype that hematopoietic stem cells have differentiated to the stage of hematopoietic stem cells. ) and hematopoietic differentiation potential detection (CFU monoclonal colony formation).
  • Example 4 Directed differentiation of hematopoietic mesoderm in a monolayer differentiation system
  • the hiPSCs obtained with reference to Example 1 are initial cells. Spread hiPSCs into well plates coated with matrigel, VTN or LN. Hematopoietic differentiation was initiated by switching the maintenance medium to medium iHSC-A (denoted as day 0). Day 0 to day 3 of hematopoietic differentiation is the stage of mesoderm formation, and directional differentiation to Brachyury+CD56+ mesoderm is achieved ( Figure 3).
  • iHSC-A composition on the one hand DMEM:F12 (Gibco) added Glutamax (Gibco), on the other hand Neurobasal media (Gibco) supplemented with N2 (Gibco) and B27 (Gibco), and then the two were mixed in a 1:1 configuration; then additionally added 5-10 ⁇ M CHIR99021 and 5-10 ⁇ M SB 4 (iHSC-A group) or 5-10 ⁇ M CHIR99021 and 12.5-25ng/ml BMP4 (hiPSC-Pro group).
  • Hematopoietic stem cells can be efficiently obtained by continuing to induce differentiation in the iHSC-A group, while the differentiated mesoderm cells in the hiPSC-Pro group Can not be effectively differentiated to obtain hematopoietic cells.
  • Figure 4 detects the expression of TRA-1-85, SSEA4, and CD56 in mesoderm cells induced and differentiated by medium iHSC-A.
  • Example 5 Induction of differentiation of hematopoietic endothelium
  • the 3rd to 4th day of hematopoietic differentiation is the hematopoietic endothelial differentiation stage (this stage can last for 2-4 days), and the cytokine combination of VEGF combined with bFGF is used to achieve directional differentiation to CD31+ (or CD144+) CD34+CD184+ hematopoietic endothelium.
  • the hematopoietic endothelial induction stage was from day 3 to day 4, and the induction medium used was EGM 2medium (Lonza), and 50ng/ml VEGF and 10ng/ml bFGF were additionally added. After 2 days of induction, a population of endothelial cells with hematopoietic differentiation potential was obtained.
  • the mesoderm obtained by the method of the present application can be effectively differentiated into hematopoietic endothelium through various methods.
  • Example 6 Induction of differentiation of hematopoietic endothelium
  • the iHSC-B (VEGF+Forskolin) used is StemPro34 medium added with 100-200ng/ml VEGF and 2-4 ⁇ M Forskolin, and Glutamine; or iHSC-B (Deoxyshikonin+Forskolin) lin) is to add 1-6 ⁇ M Deoxyshikonin and 2-4 ⁇ M Forskolin, and add Glutamine's StemPro34 medium; or iHSC-B (Isoarnebin I+Forskolin) is to add 1-10 ⁇ M Isoarnebin I and 2-4 ⁇ M Forskolin, and add Glutamine's StemPro34 medium; or iHSC-B (Isoarnebin 4+Forskolin) skolin) is StemPro34 medium added with 100-200ng/ml VEGF and 2-4 ⁇ M Forskolin, and Glutamine; or iHSC-B (Deoxyshikonin+Forskolin) lin
  • Figure 5A is an example of iHSC-B (VEGF+Forskolin) and iHSC-B (Deoxyshikonin+Forskolin) used.
  • Figure 5B and Figure 5C illustrate the iHSC-B (VEGF+Forskolin) and iHSC-B (Deoxyshikonin+Forskolin) used.
  • Example 7 Induced differentiation of hematopoietic stem cells
  • the 5th day to the 20th day of hematopoietic differentiation is the hematopoietic stem cell differentiation stage, and the directional differentiation into CD43+CD45+ hematopoietic stem cells is achieved by combining various cytokines with the small molecule SB431542.
  • the hematopoietic stem cell induction stage is from the 5th day to the 20th day.
  • the culture medium used is STEMdiff APEL2 medium (Stemcell), additionally added 50ng/ml SCF (Peprotech), 50ng/ml FLT3-L (Peprotech), 50ng/ml TPO (Peprotech), 10ng/ml IL-3 (Peprotech), 10ng/ml VEGF (Peprotech) protech), 10ng/ml bFGF (Peprotech) and 10 ⁇ M SB-431542 (Selleck).
  • hematopoietic stem cells After about 6 days of induction, a large number of hematopoietic stem cells are obtained, which can be used for the later directed differentiation of immune cells (for example, CD4 T cells, CD8 T cells and NK cells, etc.). It can be seen that the hematopoietic endothelium obtained in this application can be differentiated into hematopoietic stem cells through various methods.
  • Example 8 Induced differentiation of hematopoietic stem cells
  • hematopoietic stem cell differentiation stage was started to achieve directional differentiation to CD34+CD43+CD45+ or CD43+CD45+ hematopoietic stem cells.
  • iHSC-C is StemPro34 (Gibco), additionally add 2mM GluMAX (Gibco), 100ng/ml SCF, 25ng/ml GM-CSF, 50ng/ml IL-3, 0-100ng/ml IL-6, 50-100ng/ml ml FLT-3L, 50-100ng/ml TPO, 25-50ng/ml ascorbic acid, 5-10 ⁇ M Resveratrol, 5-10 ⁇ M SB431542, and an additional 1-2 ⁇ M UM729 can be added.
  • hematopoietic stem cells After about 5-10 days of induction (sustainable induction and multiple collections, until no remaining non-hematopoietic stem cells remain in the well), a large number of hematopoietic stem cells were obtained (Figure 6) , can be used for the later directed differentiation of immune cells (for example, CD4 T cells, CD8 T cells and NK cells, etc.).
  • Hematopoietic endothelial cells derived from a variety of induction differentiation methods can be cultured with iHSC-C to obtain hematopoietic stem cells.
  • the hematopoietic stem cells induced and differentiated by the medium iHSC-C can be effectively differentiated into T cells and NK cells through various methods.
  • Example 9 Induced differentiation of hematopoietic stem cells
  • hematopoietic mesoderm After obtaining hematopoietic mesoderm, continue to induce differentiation of hematopoietic endothelium.
  • the medium was switched to iHSC-D, and the hematopoietic stem cell differentiation stage was started to achieve directional differentiation to CD34+CD43+CD45+ or CD43+CD45+ hematopoietic stem cells.
  • the induction stage of hematopoietic stem cells is from the 3rd day to the 20th day (the collection of hematopoietic stem cells can last up to about 20 days, and can be collected multiple times).
  • the iHSC-D basic medium is StemPro34 (Gibco), additionally added 2mM GluMAX (Gibco), 25-50ng/ml ascorbic acid; or the iHSC-D basic medium is MyeloCult TM H5100 (stemcell), additionally added 25-50 ng/ml ascorbic acid; or add 25-50ng/ml ascorbic acid to StemSpan TM SFEM II for iHSC-D minimal medium.
  • iHSC-D(VEGF) comprising the minimal medium above was also supplemented with 100-200 ng/ml VEGF, 2-4 ⁇ M forskolin, 5-10 ⁇ M Resveratrol and 5-10 ⁇ M SB431542.
  • iHSC-D (Deoxyshikonin) comprising the above minimal medium was also supplemented with 1-6 ⁇ M Deoxyshikonin, 2-4 ⁇ M forskolin, 5-10 ⁇ M Resveratrol and 5-10 ⁇ M SB431542.
  • the iHSC-D (Isoarnebin I) including the above minimal medium were also supplemented with 1-10 ⁇ M Isoarnebin I, 2-4 ⁇ M forskolin, 5-10 ⁇ M Resveratrol and 5-10 ⁇ M SB431542, respectively.
  • the iHSC-D (Isoarnebin 4) including the above minimal medium were also supplemented with 1-10 ⁇ M Isoarnebin 4, 2-4 ⁇ M forskolin, 5-10 ⁇ M Resveratrol and 5-10 ⁇ M SB431542, respectively.
  • hematopoietic stem cells ( Figure 7) are obtained, which can be used for the later directed differentiation of immune cells (for example, CD4T cells, CD8T cells and NK cells, etc.).
  • Hematopoietic-related mesoderm cells derived from a variety of induced differentiation methods can be cultured with iHSC-D to obtain hematopoietic stem cells.
  • Hematopoietic stem cells induced by medium iHSC-D can be effectively differentiated into T cells and NK cells through various methods.
  • the amount of hiPSCs increased from a low plating density of approximately 2.5x10 4 cells/cm 2 to a high The plating density was about 10x10 4 cells/cm 2 /well, and hematopoietic differentiation was detected (Fig. 8).
  • hiPSCs were tested for hematopoietic differentiation by iHSC platform 1 ( Figure 9); hiPSCs were tested for hematopoietic differentiation by iHSC platform 2 ( Figure 10).
  • the hematopoietic potential of the obtained iHSCs was detected by CFU monoclonal colony formation assay.
  • the obtained iHSCs were mixed using MethoCult H4034 Optimum (STEMCELL Technologies) mixed semi-solid medium. The mixture was then transferred to a well plate, incubated at 37°C, 5% CO 2 , and 100% humidity for 14 days, and then observed for colonies.
  • Each colony was classified according to its morphology (as shown in Figure 11), and it was observed and recorded that the appearance of multiple lineage clone types such as CFU-GM, CFU-G, CFU-MK, BFU-E, and CFU-GEMM indicated that the iHSC had a relatively omnipotent hematopoietic differentiation ability.
  • Example 13 Continued differentiation of specific hematopoietic lineages after differentiation using iHSC culture platform
  • Sorted or enriched iHSC cells are further differentiated into various specific cell types along the hematopoietic lineage, including T cells and NK cells.
  • Exemplary iHSCs were further differentiated into T cells.
  • the enriched iHSC cells were added to well plates coated with vitaminectin and hDLL-4/Fc, and the incubation medium composition: StemSpan SFEM II, 100 ⁇ ITS-G (1 ⁇ ), 55 ⁇ M 2-Mercaptoethanol, 50 ⁇ g/ml Vc, Glutamax 2Mm, 50ng/ml rHSCF, 50ng/ml rhTPO, 50ng/ml rhIL-7, 50ng/ml FLT3L, 30nM rhSDF-1 ⁇ and 15 ⁇ M SB203580, culture for about 28 days, transfer the cells into a new coated well plate, add the medium composition: StemSpan SFEM II, 1 ⁇ ITS-G, 50 ⁇ g/ml ascorbic acid-2-phosphate 1 ⁇ PSG, 10ng/ml rhIL-7, 10ng /ml rhIL-2, and 10nM dexamethasone, continued to culture for 14 days.
  • Exemplary iHSCs were further differentiated into NK cells.
  • the enriched iHSC cells were co-cultured with OP9-DL1 cells.
  • the medium was StemSpan SFEM II, 300IU/mL IL-2, 50ng/mL IL-12, 50ng/ml IL-18, 50ng/ml IL-21, 50ng/ml IL27, 50ng/ml IL-15, 5% human platelet lysate, 5mmol/L nicotinamide (NAM), 5 mg/ml Vitamin E and 5 ⁇ g/mL Heparin, cultivated for about 38 days.
  • Cells were collected for CD45/CD34/CD56/CD16 flow staining to detect the proportion of NK cells. The results show that the proportion of NK cells can reach up to more than 95%.

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种将多能干细胞分化为造血细胞谱系的培养平台、细胞培养基和方法。使用该培养平台和方法产生的多能干细胞来源的造血细胞谱系，能够无血清、无滋养层、单层培养且不形成拟胚体。该多能干细胞来源的造血细胞谱系包括造血内皮细胞、造血干细胞、造血多能祖细胞、造血祖细胞、T祖细胞、NK祖细胞、T细胞和NK细胞。

Description

诱导细胞分化的方法
相关申请
本专利申请要求于2022年1月20日递交的申请号为202210066079.2的中国专利申请的优先权；要求2022年8月3日递交的申请号为202210935441.5的中国专利申请的优先权；要求2022年8月4日递交的申请号为202210932654.2的中国专利申请的优先权。
技术领域
本申请涉及诱导干细胞分化的方法、培养平台和组合物，具体涉及干细胞分化为中胚层、造血内皮、造血干细胞、造血多能祖细胞、T细胞或NK细胞的方法、培养平台和组合物。
背景技术
造血干细胞(hematopoietic stem cells，HSCs)是存在于造血组织和血液中的一类成体干细胞，具有自我更新能力和分化为所有成熟血细胞的潜能。大部分恶性血液疾病，如急性髓系白血病、慢性髓性白血病的发生和进展，都直接或间接地与HSCs异常有关。自从20世纪60年代发展至今，HSCs移植已经成为治疗各型白血病、再生障碍性贫血、β-地中海贫血等严重血液病和免疫系统疾病的重要医疗手段，同时也被应用于某些实体瘤的治疗，如淋巴瘤、小细胞肺癌等。
目前临床应用的HSCs有三大来源，即骨髓、外周血和脐带血，天然来源造血干细胞数量极其不足，是限制其临床应用的主要障碍。研究人员也有开发从胚胎干细胞或诱导多能干细胞分化为造血干细胞的方法，其中，需要经历具有造血分化潜力的中胚层细胞的培养。
现有研究的造血分化方法中，被广泛认可的具有造血分化潜力的中胚层分化方法有限，主要有：1)Activin A、BMP4、VEGF和bFGF；2)CHIR99021、BMP4、Activin A(J Cell Physiol.2020August 10.)等，均需添加多个细胞因子。由于细胞因子制备成本高；且作为生物活性蛋白，易降解；用在工业上，不同批次细胞因子活性差异大，不稳定，且需新鲜添加；这都导致包含细胞因子的造血分化方法稳定性差、转化应用困难。
发明内容
鉴于此本申请建立一种安全稳定、高效、低成本的干细胞诱导分化成中胚层细胞、造血内皮、造血干细胞、造血多能祖细胞、T细胞或NK细胞的分化方法、培养平台及其组合物。
本申请提供了一种新颖的，使该细胞快速高效的分化方法。具体技术方案如下：
在本申请的第一方面，提供了一种诱导干细胞分化的方法，在中胚层诱导阶段使多能干细胞与中胚层分化诱导剂接触以产生中胚层细胞，所述中胚层分化诱导剂包括GSK-3 抑制剂\Wnt通路激活剂、以及式I所示的化合物A，
其中，R1选自：
进一步的，所述中胚层分化诱导剂还包括低氧诱导因子(HIF)活化剂；优选的，所述HIF活化剂是HIF-1a活化剂。
进一步的，所述GSK-3抑制剂\Wnt通路激活剂是小分子化合物。
进一步的，所述式I所示的化合物中，X1选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
进一步的，所述式I所示的化合物中，X2选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
进一步的，所述式I所示的化合物中，X3选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
进一步的，所述式I所示的化合物中，R2选自：
在一优选实施例中，所述X4、X5、X6、或X7选自H、甲基、乙基、羟基、甲氧基、乙氧基、或卤素，X4、X5、X6、或X7可以相同，也可以不同；
优选的，所述R2选自以下任一结构：
进一步的，所述的HIF活化剂是小分子化合物。
进一步的，所述中胚层细胞的CD56或T基因显示阳性。
进一步的，所述GSK-3抑制剂\Wnt通路激活剂选自CHIR99021、SB216763、CHIR-98014、LY2090314、BIO-acetoxime、Alsterpaullone、CP21R7。
进一步的，所述的HIF活化剂是脯氨酰羟化酶2抑制剂。
进一步的，所述的HIF活化剂选自Roxadustat、IOX2、DMOG、PT-2385、Belzutifan、PX-478、LW6、Oltipraz、Daprodustat、BAY87-2243、KC7F2、Molidustat、PT2399、Amifostine、Enarodustat、Chlorogenic acid、ML228、Acriflavine、Vadadustat、FG-2216、Tilorone dihydrochloride、Fraxinellone、1,4-DPCA、MK-8617、Glucosamine hydrochloride、Hydralazine hydrochloride、AKBA。
进一步的，在前述任一所述的方法中，所述干细胞分化的体系中不含血清或血清替代物。
进一步的，在前述任一所述的方法中，所述干细胞分化体系中不含外源添加的细胞；
进一步的，在前述任一所述的方法中，所述的外源添加的细胞是滋养层细胞或共培养细胞。
进一步的，在前述任一所述的方法中，所述干细胞分化体系是单层分化体系。
进一步的，在前述任一所述的方法中，所述干细胞是人胚胎干细胞(hESC)或人诱导多能干细胞(hiPSC)。
进一步的，在前述任一所述的方法中，所述方法不形成内胚层细胞或外胚层细胞。
进一步的，在前述任一所述的方法中，所述中胚层分化诱导剂不包含BMP4。
进一步的，在前述任一所述的方法中，所述铺板密度是不高于1*104cells/cm2；优选的，所述铺板密度不高于5000cells/cm2。
进一步的，在前述任一所述的方法中，所述铺板密度是不低于1000cells/cm2；更优选的，所述铺板密度为1500-5000cells/cm2。
进一步的，在前述任一所述的方法中，所述铺板密度是不低于1*104cells/cm2；优选的，所述铺板密度为1*104cells/cm2-5*104cells/cm2；更优选的，所述铺板密度为3.7x104-4.7x104cells/cm2。
进一步的，在前述任一所述的方法中，所述铺板密度是1000cells/cm2-5*104cells/cm2。
进一步的，在前述任一所述的方法中，所述GSK-3抑制剂\Wnt通路激活剂的添加浓度是0.1-20μM。
进一步的，在前述任一所述的方法中，所述式I所示的化合物的添加浓度是0.1-20μM。
进一步的，在前述任一所述的方法中，所述式I所示的化合物为：
进一步的，在前述任一所述的方法中，所述中胚层细胞是造血相关中胚层细胞。
进一步的，在前述任一所述的方法中，所述GSK-3抑制剂\Wnt通路激活剂为CHIR99021。
进一步的，在前述任一所述的方法中，所述中胚层分化诱导剂不包含Activin A。
进一步的，本申请还提供了前述任一所述的方案制备的中胚层细胞的用途，用于经 进一步诱导分化制备造血内皮细胞、造血干细胞、造血干祖细胞、造血前体细胞以及免疫细胞；优选的，所述免疫细胞包含T细胞、NK细胞、B细胞、或巨噬细胞。
进一步本申请还提供以下技术方案：
项1.引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血内皮细胞引导阶段使多能干细胞来源的造血相关中胚层细胞接触包含高浓度VEGF或VEGF通路激活剂的组合物，以获得造血内皮细胞类群。
项2.如项1所述的方法，其特征在于，所述组合物包含30-200ng/ml VEGF，优选地，50-200ng/ml VEGF，更优选地，100-200ng/ml VEGF。
项3.引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血内皮细胞引导阶段使多能干细胞来源的造血相关中胚层细胞接触包含VEGF通路激活剂的组合物，以获得造血内皮细胞类群。
项4.如项1-3任一所述的方法，其特征在于，所述VEGF通路激活剂包括紫草醌衍生物；优选的，所述紫草醌衍生物包括arnebin以及其结构类似物。
项5.如项1-4任一所述的方法，其特征在于，所述VEGF通路激活剂包括Deoxyshikonin、Isoarnebin I或其组合。
项6.如项1-5任一所述的方法，其特征在于，所述组合物还包括cAMP通路激活剂。
项7.如项5或6所述方法，其特征在于，所述组合物包括1-10μM Deoxyshikonin；优选地，1-6μM Deoxyshikonin，更优选地，2-6μM Deoxyshikonin。
项8.如项5或6所述方法，其特征在于，所述组合物包括1-20μM Isoarnebin I，优选地，5-15μM Isoarnebin I，更优选地，8-15μM Isoarnebin I。
项9.如项6-8任一所述方法，其特征在于，所述cAMP通路激活剂包括Forskolin。
项10.如项9所述的方法，其特征在于，所述组合物包括1-20μM Forskolin；优选地，2-10μM Forskolin更优选地，2-4μM Forskolin。
项11.如项1-10任一所述的方法，其特征在于，所述组合物还包括StemPro34。
项12.如项1-11任一所述的方法，其特征在于，在中胚层细胞引导阶段是iPSC接触包含化合物A的另一组合物，以获得所述造血相关中胚层细胞；所述另一组合物包括GSK-3抑制剂\Wnt通路激活剂、以及式I所示的化合物A，
其中，R1选自：
项13.如项12所述的方法，其特征在于，所述另一组合物还包括低氧诱导因子(HIF)活化剂；优选的，所述HIF活化剂是HIF-1a活化剂。
项14.如项12所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂是小分子化合物。
项15.如项12所述的方法，其特征在于，所述式I所示的化合物中，X1选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
项16.如项12所述的方法，其特征在于，所述式I所示的化合物中，X2选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
项17.如项12所述的方法，其特征在于，所述式I所示的化合物中，X3选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
项18.如项12所述的方法，其特征在于，所述式I所示的化合物中，R2选自：
项19.如项18所述的方法，其特征在于，所述X4、X5、X6、或X7选自H、甲基、乙基、羟基、甲氧基、乙氧基、或卤素，X4、X5、X6、或X7可以相同，也可以不同；
优选的，所述R2选自以下任一结构：
项20.如项13所述的方法，其特征在于，所述的HIF活化剂是小分子化合物。
项21.如项12所述的方法，其特征在于，所述中胚层细胞的CD56或T基因显示阳性。
项22.如项14所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂选自CHIR99021、SB216763、CHIR-98014、LY2090314、BIO-acetoxime、Alsterpaullone、CP21R7。
项23.如项20所述的方法，其特征在于，所述的HIF活化剂是脯氨酰羟化酶2抑制剂。
项24.如项20所述的方法，其特征在于，所述的HIF活化剂选自Roxadustat、IOX2、DMOG、PT-2385、Belzutifan、PX-478、LW6、Oltipraz、Daprodustat、BAY87-2243、KC7F2、Molidustat、PT2399、Amifostine、Enarodustat、Chlorogenic acid、ML228、Acriflavine、Vadadustat、FG-2216、Tilorone dihydrochloride、Fraxinellone、1,4-DPCA、MK-8617、Glucosamine hydrochloride、Hydralazine hydrochloride、AKBA。
项25.如项1-24任一所述的方法，其特征在于，所述多能干细胞向造血细胞系的分化 处于无滋养层条件下。
项26.项1-25任一所述的方法，其特征在于，所述多能干细胞向造血细胞系的分化处于无基质条件或有基质条件下。
项27.项26所述的方法，其特征在于，所述基质包括matrigel、VTN-N、laminin-511、或laminin-521。
项28.如项1-27任一所述的方法，其特征在于，所述组合物和另一组合物不包含bFGF、BMP4或Activin A。
项29.如项1-28任一所述的方法，其特征在于，所述多能干细胞向造血细胞系的分化是单层分化体系。
项30.如项12-29任一所述的方法，其特征在于，所述铺板密度是不高于1*104cells/cm2；优选的，所述铺板密度不高于5000cells/cm2。
项31.如项1-30任一所述的方法，其特征在于，所述铺板密度是不低于1000cells/cm2；更优选的，所述铺板密度为1500-5000cells/cm2。
如项1-31任一所述的方法，其特征在于，所述铺板密度是不低于1*104cells/cm2；优选的，所述铺板密度为1*104cells/cm2-5*104cells/cm2；更优选的，所述铺板密度为3.7x104-4.7x104cells/cm2。
项33.如项12-32任一所述的方法，其特征在于，所述铺板密度是1000cells/cm2-5*104cells/cm2。
项34.如项12-33任一所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂的添加浓度是0.1-20μM。
项35.如项12-34任一所述的方法，其特征在于，所述式I所示的化合物的添加浓度是0.1-20μM。
项36.如项12-35任一所述的方法，其特征在于，所述式I所示的化合物选自：
项37.如项12-36任一所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂为CHIR99021。
项38.项1-37任一所述的血内皮细胞类群的用途，用于经进一步引导分化制备造血内皮细胞、造血干细胞、造血祖细胞、造血前体细胞以及免疫细胞；优选的，所述免疫细胞包含T细胞、NK细胞、B细胞、或巨噬细胞。
进一步，本申请还涉及如下内容：
(1).引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血干细胞引导阶段使多能干细胞来源的造血相关中胚层细胞或造血内皮细胞(HE)接触组合物I，以获得造血干细胞；所述组合物I包含：Notch信号通路激活剂和TGFβ受体/ALK5抑制剂。
(2).引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血干细胞诱导阶 段使多能干细胞来源的造血内皮细胞接触组合物II，以获得造血干细胞；所述组合物II包含：Notch信号通路激活剂、TGFβ受体/ALK5抑制剂和造血干细胞促增殖分子。
(3).如(1)或(2)所述的方法，其特征在于，所述Notch信号通路激活剂包括Resveratrol和/或VPA。
(4).如(1)-(3)任一所述的方法，其特征在于，所述TGFβ受体/ALK抑制剂包括SB431542、E-616452和/或A83-01。
(5).如(1)-(4)任一所述的方法，其特征在于，所述组合物I或组合物II中的Resveratrol的添加浓度是1-20μM；优选地，5-15μM，更优选地，6-12μM。
(6).如(1)-(5)任一所述的方法，其特征在于，所述组合物I或组合物II中的SB431542的添加浓度是1-20μM；优选地，5-15μM，更优选地，6-12μM。
(7).如(2)-(6)任一所述的方法，其特征在于，所述造血干细胞促增殖分子包括UM729、UM171、SR1或其组合。
(8).如(2)-(7)任一所述的方法，其特征在于，所述组合物I或组合物II中的UM729的添加浓度是0.1-5μM UM729；优选地，0.5-4μM，更优选地，0.5-2μM。
(9).如(2)所述的方法，其特征在于，所述组合物II中的Resveratrol的添加浓度是6-12μM、SB431542的添加浓度是6-12μM SB431542、和UM729的添加浓度是0.5-2μM。
(10).如(1)-(9)任一所述的方法，其特征在于，所述方法还包括：所述多能干细胞接触组合物B，以获得所述造血内皮细胞；所述组合物B包含高浓度VEGF和/或VEGF通路激活剂。
(11).如(10)所述的方法，其特征在于，所述组合物B中的VEGF的添加浓度是30-200ng/ml，优选地，50-200ng/ml，更优选地，100-200ng/ml。
(12).如(10)或(11)所述的方法，其特征在于，所述VEGF通路激活剂包括紫草醌衍生物；优选的，所述紫草醌衍生物包括arnebin以及其结构类似物。
(13).如(9)-(11)任一所述的方法，其特征在于，所述VEGF通路激活剂包括Deoxyshikonin、Isoarnebin I或其组合。
(14).如(10)-(13)任一所述的方法，其特征在于，所述组合物B还包括cAMP通路激活剂。
(15).如(10)-(14)所述方法，其特征在于，所述组合物B中的Deoxyshikonin的添加浓度是1-10μM；优选地，1-6μM，更优选地，1-5μM。
(16).如(10)-(15)任一所述方法，其特征在于，所述组合物B中的Isoarnebin I的添加浓度是1-20μM，优选地，5-15μM，更优选地，8-15μM。
(17).如(10)-(16)任一所述方法，其特征在于，所述cAMP通路激活剂包括Forskolin。
(18).如(10)-(17)任一所述的方法，其特征在于，所述组合物B中的Forskolin的添加浓度是1-20μM；优选地，2-10μM；更优选地，2-4μM。
(19).如(10)所述方法，其特征在于，所述组合物B中的Deoxyshikonin的添加浓度是 1-5μM、Isoarnebin I的添加浓度是8-15μM、和Forskolin的添加浓度是2-4μM。
(20).如(1)-(19)任一所述的方法，其特征在于，所述组合物I、组合物II或组合物B，还包括StemPro34。
(21).如(20)所述的方法，其特征在于，所述组合物I或组合物II还包括ascorbic acid,SCF,IL-3,FLT-3L,GM-CSF,IL-6,TPO。
(22).如(1)-(21)任一所述的方法，其特征在于，所述造血内皮细胞衍生自诱导多能干细胞的造血相关中胚层细胞。
(23).如(1)-(22)任一所述的造血干细胞的用途，用于经进一步诱导分化制备造血祖细胞、造血前体细胞以及免疫细胞；优选的，所述免疫细胞包含T细胞、NK细胞、B细胞、或巨噬细胞。
(24).引诱导多能干细胞向造血细胞系分化的方法，其特征在于，在造血干细胞诱导阶段使衍生自iPSC的造血相关中胚层细胞接触组合物III，以获得造血干细胞；所述组合物III包含：高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂、和TGFβ受体/ALK5抑制剂。
(25).如(24)所述的方法，其特征在于，所述组合物III还包括cAMP通路激活剂。
(26).如(25)所述的方法，其特征在于，所述cAMP通路激活剂包括Forskolin和/或PACAP 1-38。
(27).如(24)-(26)任一所述的方法，其特征在于，所述Notch信号通路激活剂包括Resveratrol和/或VPA。
(28).如(24)-(27)任一所述的方法，其特征在于，所述TGFβ受体/ALK抑制剂包括SB431542、E-616452和/或A83-01。
(29).如(24)-(28)任一所述的方法，其特征在于，所述组合物III中的Resveratrol的添加浓度是1-20μM；优选地，5-15μM，更优选地，6-12μM。
(30).如(24)-(29)任一所述的方法，其特征在于，所述组合物III中的SB431542的添加浓度是1-20μM；优选地，5-15μM，更优选地，6-12μM。
(31).如(24)-(30)任一所述的方法，其特征在于，所述组合物III中的VEGF的添加浓度是30-200ng/ml，优选地，50-200ng/ml，更优选地，100-200ng/ml。
(32).如(24)-(31)任一所述的方法，其特征在于，所述VEGF通路激活剂包括紫草醌衍生物；优选的，所述紫草醌衍生物包括arnebin以及其结构类似物。
(33).如(24)-(32)任一所述的方法，其特征在于，所述VEGF通路激活剂包括Deoxyshikonin、Isoarnebin I或其组合。
(34).如(24)-(33)任一所述的方法，其特征在于，所述组合物III中的Deoxyshikonin的添加浓度是1-10μM；优选地，1-6μM，更优选地，1-5μM。
(35).如(24)-(34)任一所述方法，其特征在于，所述组合物III中的Isoarnebin I的添加浓度包括1-20μM，优选地，5-15μM，更优选地，8-15μM。
(36).如(24)-(35)任一所述的方法，其特征在于，所述组合物III中的Forskolin的添加浓度是1-20μM；优选地，2-10μM；更优选地，2-4μM。
(37).如(24)-(36)任一所述的方法，其特征在于，所述组合物III包括：VEGF、Deoxyshikonin、Isoarnebin I或其组合，还包括Resveratrol，Forskolin，和SB431542。
(38).如(24)所述的方法，其特征在于，所述组合物III中的VEGF的添加浓度是100-200ng/ml或Deoxyshikonin添加浓度是1-5μM或Isoarnebin添加浓度是8-15μM，Resveratrol的添加浓度是6-12μM，Forskolin的添加浓度是2-4μM，和SB431542的添加浓度是6-12μM。
(39).如(24)-(38)任一所述的方法，其特征在于，所述组合物III还包括StemPro34、MCH5100、和/或SFEM II。
(40).如(1)、(22)-(39)任一所述的方法，其特征在于，所述方法还包括：所述多能干细胞接触包含化合物A的组合物A，以获得所述造血相关中胚层细胞；所述组合物A包含GSK-3抑制剂\Wnt通路激活剂、以及式I所示的化合物A，
其中，R1选自：
(41).如(40)所述的方法，其特征在于，所述组合物A还包括低氧诱导因子(HIF)活化剂；优选的，所述HIF活化剂是HIF-1a活化剂。
(42).如(40)所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂是小分子化合物。
(43).如(40)所述的方法，其特征在于，所述式I所示的化合物中，X1选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
(44).如(40)所述的方法，其特征在于，所述式I所示的化合物中，X2选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
(45).如(40)所述的方法，其特征在于，所述式I所示的化合物中，X3选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素。
(46).如(40)所述的方法，其特征在于，所述式I所示的化合物中，R2选自：
(47).如(46)所述的方法，其特征在于，所述X4、X5、X6、或X7选自H、甲基、乙基、羟基、甲氧基、乙氧基、或卤素，X4、X5、X6、或X7可以相同，也可以不同；
优选的，所述R2选自以下任一结构：
(48).如(41)所述的方法，其特征在于，所述的HIF活化剂是小分子化合物。
(49).如(40)所述的方法，其特征在于，所述中胚层细胞的CD56或T基因显示阳性。
(50).如(40)所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂选自CHIR99021、SB216763、CHIR-98014、LY2090314、BIO-acetoxime、Alsterpaullone、CP21R7。
(51).如(48)所述的方法，其特征在于，所述的HIF活化剂是脯氨酰羟化酶2抑制剂。
(52).如(48)所述的方法，其特征在于，所述的HIF活化剂选自Roxadustat、IOX2、DMOG、PT-2385、Belzutifan、PX-478、LW6、Oltipraz、Daprodustat、BAY87-2243、KC7F2、Molidustat、PT2399、Amifostine、Enarodustat、Chlorogenic acid、ML228、Acriflavine、Vadadustat、FG-2216、Tilorone dihydrochloride、Fraxinellone、1,4-DPCA、MK-8617、Glucosamine hydrochloride、Hydralazine hydrochloride、AKBA。
(53).如(1)-(52)任一所述的方法，其特征在于，所述多能干细胞向造血细胞系的分化处于无滋养层、无血清条件下。
(54).如(1)-(53)任一所述的方法，其特征在于，所述多能干细胞向造血细胞系的分化处于有细胞外基质条件下。
(55).如(54)所述的方法，其特征在于，所述细胞外基质包括matrigel、VTN-N、laminin-511、laminin-521或其组合。
(56).如(1)-(55)任一所述的方法，其特征在于，所述任一组合物均不包含bFGF、BMP4或Activin A。
(57).如(1)-(56)任一所述的方法，其特征在于，所述多能干细胞向造血细胞系的分化是单层分化体系。
(58).如(40)-(57)任一所述的方法，其特征在于，所述多能干细胞铺板密度不低于1*104cells/cm2；优选的，所述铺板密度为1*104cells/cm2-5*104cells/cm2；更优选的，所述铺板密度为3.7x104-4.7x104cells/cm2。
(59).如(40)-(58)任一所述的方法，其特征在于，所述多能干细胞铺板密度不高于90％。
(60).如(40)-(59)任一所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂的添加浓度是0.1-20μM。
(61).如(40)-(60)任一所述的方法，其特征在于，所述式I所示的化合物的添加浓度是0.1-20μM。
(62).如(40)-(61)任一所述的方法，其特征在于，所述式I所示的化合物选自：
(63).如(40)-(62)任一所述的方法，其特征在于，所述GSK-3抑制剂\Wnt通路激活剂为CHIR99021。
(64).如(40)-(63)任一所述的中胚层细胞的用途，用于经进一步诱导分化制备造血内皮、造血干细胞、造血祖细胞、造血前体细胞以及免疫细胞；优选的，所述免疫细胞包含T细胞、NK细胞、B细胞、或巨噬细胞。
(65).引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血干细胞诱导阶段使多能干细胞来源的造血相关中胚层细胞或造血内皮细胞接触组合物I，以获得造血干细胞；所述组合物I包含：Resveratrol和/或VPA；还包括SB431542和/或A83-01。
(66).引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血干细胞诱导阶段使多能干细胞来源的造血内皮细胞接触组合物II，以获得造血干细胞；所述组合物包含：Resveratrol和/或VPA；SB431542和/或A83-01；还包括UM729。
(67).引导多能干细胞向造血细胞系分化的方法，其特征在于，在造血干细胞诱导阶段使多能干细胞来源的造血相关中胚层细胞接触组合物III，以获得造血干细胞；所述组合物III包含：高浓度VEGF或Deoxyshikonin或Isoarnebin，还包括Resveratrol,SB431542和Forskolin。
(68).引导多能干细胞向造血细胞系分化的方法，其特征在于，包含：(i)使多能干细胞接触包含GSK-3抑制剂\Wnt通路激活剂和如(62)所述的化合物A的组合物A，以获得中胚层细胞；(ii)使中胚层细胞接触包含高浓度VEGF或VEGF通路激活剂，和cAMP通路激活剂的组合物B,以获得造血内皮(HE)；以及(iii)使造血内皮细胞接触包含Notch信号通路激活剂、TGFβ受体/ALK5抑制剂和造血干细胞促增殖分子的组合物II，以获得造血干细胞。
(69).引导多能干细胞向造血细胞系分化的方法，其特征在于，包含：(i)使多能干细胞接触包含CHIR99021和如(62)所述的化合物A的组合物A，以获得中胚层细胞；(ii)使中胚层细胞接触包含高浓度VEGF或Deoxyshikonin或Isoarnebin，和Forskolin的组合物B,以获得造血内皮(HE)；以及(iii)使造血内皮细胞接触包含Resveratrol、SB431542和UM729的组合物II，以获得造血干细胞。
(70).引导多能干细胞向造血细胞系分化的方法，其特征在于，包含：(i)使多能干细胞接触包含GSK-3抑制剂\Wnt通路激活剂和如(62)所述的化合物A的组合物A，以获得中胚层细胞；(ii)使中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂、TGFβ受体/ALK5抑制剂、和cAMP通路激活剂的组合物III,以获得造血干细胞。
(71).引导多能干细胞向造血细胞系分化的方法，其特征在于，包含：(i)使多能干细胞接触包含CHIR99021和如(62)所述的化合物A的组合物A，以获得中胚层细胞；(ii)使中胚层细胞接触包含高浓度VEGF或Deoxyshikonin或Isoarnebin，Resveratrol,SB431542和 Forskolin的组合物III，以获得造血干细胞。
应理解，在本申请范围内中，本申请的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1.显示了人诱导性多能干细胞(hiPSC)向造血干细胞(iHSC)诱导分化的三阶段过程的示例性简图。
图2.显示了hiPSC向iHSC诱导分化的二阶段过程的示例性简图。
图3A显示了流式分析检测hiPSC向造血中胚层分化时CD56表达情况；图3B显示了免疫荧光检测hiPSC向造血中胚层分化时T表达情况。结果显示，在不添加其他细胞因子(如BMP4、Activin A)仅添加CHIR99021和SB 4的iHSC-A组,几乎所有细胞检测到T表达，标明该分化系统能够实现高效的造血相关中胚层定向分化。
图4A显示了在ECM为VTN-N培养条件下(图4A)，经培养基iHSC-A诱导分化得到的中胚层细胞的TRA-1-85、SSEA4、CD56表达情况，图4B显示在ECM)为matrigel培养条件下(图4B)，经培养基iHSC-A诱导分化得到的中胚层细胞的TRA-1-85、SSEA4、CD56表达情况。结果显示，在不同ECM培养条件下，以能够成功获得造血干细胞的基准前提下，泛表达基因TRA-1-85在中胚层定向分化过程中稳定高表达，多能性标志基因SSEA4表达在逐步降低，标明hiPSC正逐渐分化而失去多能性；CD56先呈现初始基准表达水平，随后表达水平降低然后又恢复甚至呈现高表达，说明在定向造血相关中胚层分化过程中，CD56在干细胞状态下普遍低表达，随着分化的进行CD56呈现先降后升，至中胚层时呈现高表达的动态变化过程。伴随SSEA4表达逐渐降低，CD56高表达，表明此阶段已经进入造血相关中胚层阶段。这说明在多种ECM条件下(包括VTN-N、matrigel等)，高效稳定的实现造血相关中胚层的定向分化，获得造血相关中胚层细胞，显示了iHSC培养平台高效的促分化效能。
图5显示了采用iHSC培养平台1分化的造血内皮的检测结果：图5A显示了分化获得的CD34+细胞类群包含CD73+的动静脉内皮细胞(AE/VE)和CD73-的造血内皮细胞(HE),图5B、图5C显示了iHSC-B分别VEGF+Forskolin或Deoxyshikonin+Forskolin组合时，在不同的ECM培养条件下，造血内皮的细胞表面标志物CD34、CD43、CD45、CD144、CD31、CD309、CD73表达情况。结果显示，iHSC培养平台1成功的实现了造血内皮细胞的定向分化；其中CD34、CD144、CD31的阳性率达到约20％～30％时，CD73的阳性率约占1/5左右；且分化获得的HE占比约80％，动静脉内皮(主要是动脉内皮)占比约20％。这表明了iHSC培养平台1实现高效的造血内皮定向分化。
图6显示了采用包括iHSC-B分别包括VEGF+Forskolin、Deoxyshikonin+Forskolin、Isoarnebin I+Forskolin组合的iHSC培养平台1分化的iHSC的检测结果：图6A、6B、6C分别 显示了CD34、CD45、CD43表达情况。结果显示，iHSC分化平台1能获得接近100％的CD43+CD45+的iHSC的定向分化。这表明了iHSC分化平台1实现高效稳定的造血干细胞诱导分化。
图7显示了采用包括iHSC-D分别是iHSC-D(VEGF)、iHSC-D(Deoxyshikonin)、iHSC-D(Isoarnebin I)的iHSC培养平台2分化的iHSC的检测结果：图7A、7B、7C分别显示了CD34、CD45、CD43表达情况。结果显示，iHSC分化平台2能获得接近100％的CD43+CD45+的iHSC的定向分化。这表明了iHSC分化平台2实现高效稳定的造血干细胞诱导分化。
图8A、8B分别显示了利用iHSC培养平台1、2的hiPSC高/低起始铺板密度定向分化中胚层细胞、造血内皮、iHSC早期阶段、iHSC晚期/iHPC早期阶段、iHPC阶段。重复进行的三组实验均能获得稳定一致的细胞表型变化，表明iHSC培养平台1、2具有极高的稳定性，对后续的转化应用方面，获得稳定一致的高质量细胞产品具有重要意义。
图9A和B显示了在不同ECM培养条件下，采用iHSC平台1定向分化iHSC(图9A),及iHSC的CD34/CD45/CD43/CD31/CD38的表达情况(图9B)。结果显示，几乎全部细胞呈CD43+CD45+，这表明在不同的ECM条件下，iHSC培养平台1均能稳定、高效分化获得iHSC。同时，由于CD38在iHSC和早期淋系都是阴性表达，在早期髓系是阳性表达，故检测结果显示CD38+髓系类型细胞的占比仅不到1％，说明此时的iHSC平台1可用于髓系分化和/或淋系分化或淋系分化。
图10A、B和C显示了在不同基本培养基、不同ECM培养条件下，采用iHSC平台2定向分化iHSC：几乎全部细胞呈CD43+CD45+。这表明在不同的ECM培养条件下，iHSC平台2能够稳定、高效地分化获得iHSC。同时，由于CD38在iHSC和早期淋系都是阴性表达，在早期髓系是阳性表达；图10B显示iHSC-D基本培养基为MyeloCult H5100的培养条件下，CD38+髓系类型细胞占比约20％，这说明该培养平台获得的iHSC有相对明显的早期髓系分化倾向，故该培养平台可用于髓系分化和/或淋系分化，或优选用于髓系分化。图10A、10B分别显示iHSC-D基本培养基为StemPro34、StemSpan SFEM II的培养条件下，CD38+髓系类型细胞的占比约0％～8％，这说明该培养平台可用于髓系分化和/或淋系分化或淋系分化。
图11显示了利用CFU单克隆形成实验检测iHSC培养平台1、2获得的iHSC具有全能的造血分化能力。
图12显示了利用流式细胞检测分析iHSC培养平台1、2获得的iHSC具备自发向髓淋系分化倾向，同样显示具备造血全谱系分化潜能。
图13显示了使用iHSC培养平台1、2获得的iHSC进行分化后能继续分化成T细胞。
具体实施方式
除非另外定义，本文使用的所有专业术语、符号和其它技术和科学术语或专有词汇旨 在具有本申请所属领域技术人员通常所理解的相同含义。在一些情况中，本文出于阐明和/或便于引用的目的对具有所常规理解的含义的术语加以进一步限定，本文中包括的此类进一步限定不应理解为表示与本领域常规理解的有实质上的差异。
本申请中提及的所有出版物，包括专利文件、学术论文和数据库，均可独立地通过引用其全文纳入本文用于。如果本文所示的定义与通过引用纳入本文的专利、公开申请和其它出版物中所示的定义不同或有其它情况下不一致，以本文所示的定义为主。
本申请中，有些请求保护的主题以范围形式呈现，应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供有范围上限和范围下限的范围中，在该范围的上限和下限之间的每个中间值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值，除非另外指出。
本申请涉及用于使干细胞向造血细胞谱系分化的方法、培养平台及组合物。更具体的，本申请提供了一种多阶段培养平台，其中在不同发育阶段的iPSC或iPSC来源的细胞能够被诱导产生造血细胞谱系，范围从中胚层、造血内皮细胞、造血干细胞、造血多能祖细胞至全分化的造血细胞，后者包括T细胞、B细胞、NKT细胞、NK细胞和B细胞。优选地，本申请的方法、培养平台及组合物通过避免形成EB或聚集体而从初始iPSC以规模化的方式产生造血细胞谱系。
本申请提供了用于培养和将干细胞分化为造血细胞命运(fate)的细胞培养条件、培养基、培养平台和方法。本申请提供了用于将干细胞分化成造血相关中胚层细胞、造血内皮、造血干细胞、造血多能祖细胞、T细胞、NK细胞的小分子和细胞因子组合的诱导分化剂及其培养平台。本申请提供了用于将干细胞分化成造血相关中胚层细胞、造血内皮、造血干细胞、造血多能祖细胞、T细胞、NK细胞的不添加细胞因子的小分子组合的诱导分化剂及其培养平台。本申请提供了在不添加血清/无滋养层条件下，且在可扩展和单层培养平台中，不需要EB形成的情况下，通过来自多能干细胞(包括hiPSC)的造血相关中胚层细胞、造血内皮、造血干细胞和造血多能祖细胞产生造血细胞谱系的方法和组合物。可以根据本申请的方法分化的细胞的范围从多能干细胞到确定为特定末端分化细胞和转分化细胞的祖细胞，各种谱系的细胞不通过多能中间体直接转化成造血细胞。类似地，通过干细胞分化产生的细胞范围从专能干细胞或祖细胞到终末分化的干细胞，以及所有中间的造血细胞谱系。
本申请提供了用于在单层培养中从多能干细胞分化和扩增造血细胞谱系的方法和组合物，其包括使多能性干细胞与GSK3β抑制剂以及小分子SB4接触，获得并扩增中胚层细胞，而不从多能干细胞形成拟胚体。然后使中胚层细胞与包含高浓度VEGF和/或VEGF通路激活剂和任选地cAMP通路激活剂接触，以获得造血内皮，而不形成拟胚体。随后使造 血内皮与Notch信号通路激活剂和TGFβ受体/ALK5抑制剂和任选地造血干细胞促增殖分子接触，获得并扩增了造血干细胞，而不形成拟胚体。
本申请提供了用于在单层培养中从多能干细胞分化和扩增造血细胞谱系的方法和组合物，其包括使多能性干细胞与GSK3β抑制剂以及小分子SB4接触，获得并扩增中胚层细胞，而不从多能干细胞形成拟胚体。然后使中胚层细胞与Notch信号通路激活剂和TGFβ受体/ALK5抑制剂，和任选地cAMP通路激活剂，和任选地高浓度VEGF和/或VEGF通路激活剂接触，获得并扩增造血干细胞，而不形成拟胚体。
本文提供的利用单层分化获得造血细胞谱系的方法优于EB介导的多能干细胞分化，因为EB的形成导致细胞分化不均一、且细胞扩增效率低下。本申请提供了不添加细胞因子的小分子组合的诱导分化剂，建立了有利于分化为造血相关中胚层、造血内皮、造血干细胞、造血多能祖细胞的单层培养平台，导致造血干细胞、造血多能祖细胞及其分化后代如T、B、NKT和NK细胞的衍生。本文的小分子组合诱导剂和单层分化策略将增强的分化效率与大规模扩增结合起来，可以递送治疗相关数量的多能干细胞来源的造血细胞谱系用于各种治疗应用。此外，本申请公开了利用小分子组合诱导分化剂和单层培养方法获得功能性造血细胞谱系，这些造血细胞谱系能进行体外分化、离体调节和实现体内长期造血自我更新、造血重建和造血移植。
本申请提供了用于获得多能干细胞来源的造血相关中胚层细胞的培养平台，其包含：第I组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增造血相关中胚层细胞。
本申请提供了用于获得多能干细胞来源的造血细胞谱系的培养平台，其包含：第II组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂，该培养基适于从中胚层细胞分化和扩增造血内皮；(iii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子，该培养基适于从造血内皮分化和扩增造血干细胞。
本申请提供了用于获得多能干细胞来源的造血细胞谱系的培养平台，其包含：第III组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂，该培养基适于从中胚层细胞分化和扩增造血干细胞。
本申请提供了用于获得多能干细胞来源的造血干细胞的培养平台，其包含：第II组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂，该培养基适于从中胚层细胞分化和扩增造血内皮；(iii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子，该培养 基适于从造血内皮分化和扩增造血干细胞。
本申请提供了用于获得多能干细胞来源造血干细胞的培养平台，其包含：第III组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂，该培养基适于从中胚层细胞分化和扩增造血干细胞。
本申请提供了用于获得多能干细胞来源的造血多能祖细胞的培养平台，其包含：第II组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂，该培养基适于从中胚层细胞分化和扩增造血内皮；(iii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子，该培养基适于从造血内皮分化和扩增造血干细胞。
本申请提供了用于获得多能干细胞来源的造血多能祖细胞的培养平台，其包含：第III组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂，该培养基适于从中胚层细胞分化和扩增造血干细胞。
在一些实例中，上述培养平台的多能干细胞是iPSC。在一些实例中，iPSC是初始iPSC。在一些实例中，上述培养平台的第I、II、III组不含BMP4、Activin A、bFGF、LIF等细胞因子成分和FBS、KOSR等血清类成分。
在上述培养平台的一些实施方式中，进一步包含附加培养基，该培养基适于接种和扩增多能干细胞。在一些实例中，第I、II或III组的(i)培养基进一步包含：一种或多种选自DMEM/F12、Neurobasal、N2、B27和β-Me。在一些实例中，第II组的(ii)培养基进一步包含：一种或多种选自StemPro34、Glutamine。在一些实例中，第II组的(iii)培养基进一步包含：一种或多种选自StemPro34、Glutamine、SCF、GM-CSF、IL-3、IL-6、FLT-3L、TPO、ascorbic acid。在一些实例中，第III组的(ii)培养基进一步包含：一种或多种选自StemPro34、Glutamine、ascorbic acid；或一种或多种选自MCH5100、ascorbic acid；或一种或多种选自SFEM II、ascorbic acid。
本申请提供了用于获得多能干细胞来源的造血内皮的培养平台，其包含：第IV组：(i)培养基，其包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂，该培养基适于从中胚层细胞分化和扩增造血内皮细胞。在一些实例中，上述培养平台的第IV组不含GSK3β抑制剂、Activin A、bFGF，其中该培养基适于接种和扩增中胚层细胞。在一些实例中，第IV组的(i)培养基进一步包含附加培养基：一种或多种选自StemPro34、Glutamine。
本申请提供了用于获得多能干细胞来源的造血干细胞的培养平台，其包含：第V组： (i)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子，该培养基适于从造血内皮分化和扩增造血干细胞。在一些实例中，上述培养平台的第V组不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂、BMP途径激活剂，其中该培养基适于接种和扩增造血内皮。在一些实例中，第V组的(i)培养基进一步包含：一种或多种选自StemPro34、Glutamine、SCF、GM-CSF、IL-3、IL-6、FLT-3L、TPO、ascorbic acid。
本申请提供了用于获得多能干细胞来源的造血干细胞的培养平台，其包含：第VI组：(i)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂，该培养基适于从中胚层细胞分化和扩增造血干细胞。在一些实例中，上述培养平台的第VI组不含bFGF、BMP途径激活剂、GSK3β抑制剂，其中该培养基适于接种和扩增中胚层细胞。在一些实例中，第VI组的(i)培养基进一步包含附加培养基：一种或多种选自StemPro34、Glutamine、ascorbic acid；或一种或多种选自MCH5100、ascorbic acid；或一种或多种选自SFEM II、ascorbic acid。
在上述培养平台的一些实例中，每组的培养基还可以任选地包含低氧诱导因子(HIF)活化剂。
本申请还提供了用于分化和扩增多能干细胞来源的造血细胞谱系的组合物，其包含一种或多种下述第I、II、III、IV、V或VI组。
第I组：(i)培养基，其包含GSK3β抑制剂和小分子SB4；以及iPSC，并适于从多能干细胞分化和扩增造血相关中胚层细胞。
第II组：(i)培养基，其包含GSK3β抑制剂和小分子SB4；以及iPSC，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂；以及中胚层细胞，该培养基适于从中胚层细胞分化和扩增造血内皮；(iii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子，以及造血内皮，该培养基适于从造血内皮分化和扩增造血干细胞。
第III组：(i)培养基，其包含GSK3β抑制剂和小分子SB4，以及iPSC，该培养基适于从多能干细胞分化和扩增中胚层细胞；(ii)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂，以及中胚层细胞，该培养基适于从中胚层细胞分化和扩增造血干细胞。
第IV组：(i)培养基，其包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂，以及中胚层细胞，该培养基适于从多能干细胞来源的中胚层细胞分化和扩增造血内皮。
第V组：(i)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子，以及造血内皮，该培养基适于从多能干细胞来源的造血内皮分化和扩增造血干细胞。
第VI组：(i)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂，以及中胚层细胞，该培养基适于从多能干细胞来源的中胚层细胞分化和扩增造血干细胞。
在用于分化和扩增多能干细胞来源的造血细胞谱系的组合物的一些实施方式中，多能干细胞是iPSC。在一些实例中，iPSC是初始iPSC。
在用于分化和扩增多能干细胞来源的造血细胞谱系的组合物的一些实例中，第I、II或III组的(i)培养基包含附加组分：一种或多种选自DMEM/F12、Neurobasal、N2、B27和β-Me。在一些实例中，第II组的(ii)、第IV组的(i)培养基包含附加组分：一种或多种选自StemPro34、Glutamine。在一些实例中，第II组的(iii)培养基包含附加组分：一种或多种选自StemPro34、Glutamine、SCF、GM-CSF、IL-3、IL-6、FLT-3L、TPO、ascorbic acid。在一些实例中，第III组的(ii)、V组的(i)、第VI组的(i)培养基包含附件组分：一种或多种选自StemPro34、Glutamine、ascorbic acid；或一种或多种选自MCH5100、ascorbic acid；或一种或多种选自SFEM II、ascorbic acid。
在上述用于分化和扩增多能干细胞来源的造血细胞谱系的组合物的一些实施方式中，第(I)、(II)、(III)、(IV)、(V)、(VI)组另外包含：(i)培养基，其包含一种或多种选自SCF、Flt3L、IL7、IGF、IL2、IL3和IL6的生长因子和细胞因子；以及一种或多种Notch途径激活剂；以及多能干细胞来源的T祖细胞，其中该培养基不含BMP激活剂，并适于将多能干细胞来源的T祖细胞分化为T细胞，或(ii)培养基，其包含BMP激活剂，一种或多种选自SCF、Flt3L、IL7、IL2、IL3和IL6的生长因子和细胞因子；一种或多种Notch途径激活剂；以及多能干细胞来源的iHSC，其中该培养基适于将多能干细胞来源的iHSC分化为T祖细胞；且这些附加培养基适于产生多能干细胞来源的T细胞系。
本申请还提供了一种用于引导多能干细胞分化为中胚层细胞的方法，其包括：(i)使多能干细胞接触包含GSK3β抑制剂和小分子SB4的组合物，以启动从多能干细胞到造血相关中胚层细胞的分化和扩增。
一种用于引导多能干细胞分化为造血细胞谱系的方法，其包括：(i)使多能干细胞接触包含GSK3β抑制剂和小分子SB4的组合物，以启动从多能干细胞到中胚层细胞的分化和扩增；(ii)使中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂的组合物，以启动从中胚层细胞到造血内皮的分化和扩增；以及(iii)使造血内皮接触包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子的组合物，以启动从造血内皮到的造血干细胞的分化和扩增。
一种用于引导多能干细胞分化为造血细胞谱系的方法，其包括：(i)使多能干细胞接触包含GSK3β抑制剂和小分子SB4的组合物，以启动从多能干细胞到中胚层细胞的分化和扩增；以及(ii)使中胚层细胞接触包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF和/或VEGF通路激活剂的组合物，以启动从中胚层细胞到造血干细胞的分化和扩增。
一种用于引导多能干细胞分化为造血内皮的方法，其包括：(i)使多能干细胞来源的中胚层细胞接触包含高浓度VEGF或VEGF通路激活剂以及可选的cAMP通路激活剂的组合物，以启动从中胚层细胞到造血内皮的分化和扩增。
一种用于引导多能干细胞分化为造血干细胞的方法，其包括：(i)使多能干细胞来源的造血内皮接触包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子的组合物，以启动从造血内皮到造血干细胞的分化和扩增。
一种用于引导多能干细胞分化为造血干细胞的方法，其包括：(i)使多能干细胞来源的中胚层细胞接触包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF或VEGF通路激活剂的组合物，以启动从中胚层细胞到造血干细胞的分化和扩增。
上述用于引导多能干细胞分化为造血细胞谱系的方法中，多能干细胞向造血细胞谱系的分化不产生拟胚体且为单层培养形式。在一些实例中，使多能干细胞、中胚层细胞、造血内皮、造血干细胞和/或造血多能祖细胞处于低氧条件下、或添加小分子激活细胞低氧途径。在一些实例中，多能干细胞是iPSC。在一些实例中，iPSC是初始iPSC。在一些实例中，获得的中胚层细胞是Brachyury+CD56+。在一些实例中，获得的造血内皮CD31+CD34+CD184+。在一些实例中，获得的造血内皮CD31+CD144+CD34+CD184+。在一些实例中，获得的造血内皮CD144+CD34+CD184+。在一些实例中，获得的造血干细胞CD34+CD43+CD45+或造血干细胞CD43+CD45+。在一些实例中，获得的造血多能祖细胞CD34+CD43+CD45+或造血多能祖细胞CD43+CD45+。
本申请提供了一种用于产生多能干细胞来源的造血干细胞的方法，包括：(i)使iPSC接触包含GSK3β抑制剂和小分子SB4的组合物，以启动从多能干细胞向中胚层细胞的分化和扩增；(ii)使中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂以及可选的cAMP通路激活剂的组合物，以启动从中胚层细胞向造血内皮的分化和扩增；(iii)使造血内皮接触包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂、以及可选的造血干细胞促增殖分子的组合物，以启动从造血内皮向造血干细胞的分化和扩增；以及可选的，使接种的多能干细胞、中胚层细胞和/或造血内皮处于约2％至约10％的低氧张力下。在一些实例中，上述产生多能干细胞来源的造血干细胞的方法进一步包括：使iPSC接触包含ROCK抑制剂的维持培养基中，以接种和扩增iPSC；和/或其中iPSC是初始iPSC。在一些实例中，上述使iPSC分化为造血干细胞的方法不产生拟胚体且为单层培养。
本申请提供了一种用于产生多能干细胞来源的造血干细胞的方法，包括：(i)使iPSC接触包含GSK3β抑制剂和小分子SB4的组合物，以启动从多能干细胞向中胚层细胞的分化和扩增；(ii)使中胚层细胞接触包含Notch信号通路激活剂和TGFβ受体/ALK5抑制剂、以及可选的cAMP通路激活剂、以及可选的高浓度VEGF或VEGF通路激活剂的组合物，以启动从中胚层细胞向造血干细胞的分化和扩增；以及可选的，使接种的多能干细胞、中胚层细胞处于约2％至约10％的低氧张力下。在一些实例中，上述产生多能干细胞来源的造血干 细胞的方法进一步包括：使iPSC接触包含ROCK抑制剂维持培养基中，以接种和扩增iPSC；和/或其中iPSC是初始iPSC。在一些实例中，上述使iPSC分化为造血干细胞的方法不产生拟胚体且为单层培养形式。
本申请提供了一种组合物或一种多多种细胞群，包含：一种或多种产生自本文公开的培养平台、本文所述方法产生的的细胞群：多能干细胞来源的(i)Brachyury+CD56+中胚层细胞，且能够分化为造血内皮、造血干细胞、造血多能祖细胞、T祖细胞、NK祖细胞、T细胞和NK细胞；(ii)CD31+(或CD144)+CD34+CD73-CD43-CD184+造血内皮；(iii)CD43+CD45+造血干细胞；(iv)T祖细胞，其中T祖细胞是CD34+CD7+；(v)T细胞，其中T细胞是CD4+或CD8+；(vi)NK祖细胞，其中NK祖细胞是CD56+CD7+CD161+；以及(vii)NK细胞，其中NK细胞是CD56+CD57+CD16+CD94-。
本申请提供了使用所公开的方法产生的一种或多种细胞群、细胞系或克隆细胞促进造血自我更新、重建或移植的方法：多能干细胞来源的(i)Brachyury+CD56+中胚层细胞，且能够分化为造血内皮细胞、造血干细胞、T祖细胞、NK祖细胞、T细胞和NK细胞；(ii)CD31+(或CD144)+CD34+CD73-CD43-CD184+造血内皮细胞；(iii)CD43+CD45+造血干细胞；(iv)T祖细胞，其中T祖细胞是CD34+CD7+；(v)T细胞，其中T细胞是CD4+或CD8+；(vi)NK祖细胞，其中NK祖细胞是CD56+CD7+CD161+；以及(vii)NK细胞，其中NK细胞是CD56+CD57+CD16+CD94-。
本申请提供了能够使多能干细胞单层直接分化而不从多能干细胞产生拟胚体的方法和组合物，从而能够进行中胚层细胞、造血内皮细胞、造血干细胞、造血多能祖细胞的分化和扩增，其中其它造血细胞谱系能够以非常高的效率水平以规模化的、可靠的模式获得。
在本申请的任何方案中，如果提及高浓度的VEGF或VEGF通路激活剂时，高浓度是指可以浓度达到30ng/mL以上的浓度，例如可以为30-500ng/mL的浓度，例如可以为10-500ng/mL的浓度，例如可以为30-30ng/mL，例如该浓度可以为20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL、200ng/mL、210ng/mL、220ng/mL、230ng/mL、240ng/mL、250ng/mL、260ng/mL、270ng/mL、280ng/mL、290ng/mL、300ng/mL、310ng/mL、320ng/mL、330ng/mL、340ng/mL、350ng/mL、360ng/mL、370ng/mL、380ng/mL、390ng/mL、400ng/mL、410ng/mL、420ng/mL、430ng/mL、440ng/mL、450ng/mL、460ng/mL、470ng/mL、480ng/mL、490ng/mL、500ng/mL。
在本申请的任何方案中，如果提及低氧条件是指处于约2％至约10％的低氧张力下，例如可以为2％、3％、4％、5％、6％、7％、8％、9％或10％氧气浓度条件下。
如本文使用的，术语“约”或“大约”指与参考数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度相比较，改变多达15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或 长度。在一些实例中，术语“约”或“大约”指围绕参考数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度±15％、±10％、±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％或±1％的数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的，术语“基本上”或“大体上”指与参考数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度相比较，约90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度。在一些实例中，术语“大体上相同”或“基本上相同”指与参考数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度大约相同的数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的，术语“基本上不含”和“大体上不含”可互换使用，并且当用于描述组合物例如细胞群或培养基时，指不含指定物质或其来源，例如95％不含、96％不含、97％不含、98％不含、99％不含指定物质或其来源的组合物，或通过常规手段测量是无法检测的。术语在组合物中“不含”或“基本上不含”某种成分或物质还表示该成分或物质(1)没有以任何浓度包含在组合物中，或(2)在组合物中具有惰性功能，但以低的浓度存在。类似含义可应用于术语“不存在”，当指不存在组合物的特定物质或其来源时。
如本文使用的，术语“可评估的”指通过一种或多种标准方法可容易检测的数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度范围。术语“不可评估的(not appreciable)”和“不可评估的(not appreciable)”以及等价物指通过标准方法无法容易检测或无法检测的数量、水平、值、数目、频率、百分比、尺度、大小、量、重量或长度范围。在一些实例中，如果事件以小于5％、4％、3％、2％、1％、0.1％、0.01％、0.001％或更少的次数(time)发生，则它是不可评估的。
本说明书自始至终，除非上下文另有要求，否则术语“包含”应理解为暗示包括所述步骤或元件或者步骤或元件组，但不排除任何其他步骤或元件或者步骤或元件组。在特定实施方式中，术语“包括”、“具有”、“含有”和“包含”同义使用。
“由……组成”意指包括并限制于在短语“由……组成”后的任何内容。因此，短语“由……组成”指示所列出的元件是必需或强制性的，并且可不存在其他元件。
术语“T基因”，是由Brachury编码的组织特异性转录因子。
干细胞分化的体系中人多能干细胞(包括人诱导多能干细胞和人胚胎干细胞)单层定向造血相关中胚层分化体系或多层分化体系。
术语“中胚层”是指在早期胚胎发生期间出现的三个胚层中的一个，其产生各种特殊的细胞类型，包括循环系统的血细胞，肌肉，心脏，真皮，骨骼和其他支持性和结缔组织。
术语“造血细胞谱”和“造血细胞谱系”指造血发育或造血分化过程中，涉及的全部类型的造血相关细胞类群，包括和不限于造血内皮、造血干细胞、造血多能祖细胞、造血祖细胞(淋系祖细胞和髓系祖细胞等)、双阴性T祖细胞、双阳性T祖细胞、T祖细胞、 NK祖细胞、T细胞和NK细胞。
术语“EHT(endothelial to hematopoietic transition)”，即内皮-造血转换，由早期造血内皮细胞(HECs)转化为造血干细胞(HSPCs)的过程。在此过程中往往伴随早期动脉内皮细胞的存在，其对造血内皮向造血干细胞的转化，具有促进作用；同样在体外分化过程中，少量动脉内皮细胞的存在能够大大提高向造血干细胞分化的效率。
术语“造血内皮”或“永久造血内皮”(HE，Hemogenic Endothelium)或“多能干细胞来源的永久造血内皮”(iHE)指在称为内皮细胞-造血转变的过程中产生造血干细胞和造血祖细胞的内皮细胞亚群。
术语“造血干细胞(Hemopoietic stem cell，HSC)”是指尚未发育成熟的细胞，是所有造血细胞和免疫细胞的起源，它不仅可以分化为红细胞、白细胞和血小板，还可跨系统分化为各种组织器官的细胞，具有自我更新、多向分化和归巢(即定向迁移至造血组织器官)潜能。其能够产生成熟骨髓和淋巴细胞类型，包括T细胞、自然杀伤细胞和B细胞。在一些实例中，造血干细胞包括造血干细胞和祖细胞(HSPCs)库。在一些实例中，造血干细胞和祖细胞(HSPCs)库可包括三种类型的细胞：长期造血干细胞(LT-HSC)、短期造血干细胞(ST-HSPC)和造血多能祖细胞(MPP)。本申请中，造血干细胞HSC与造血祖细胞的主要表达标志物如下：HSC：Lin-CD34+CD43+CD45+CD38-CD45RA-CD90+CD49f+；LT-HSC：CD34-、CD38-、SCA-1+、Thy1.1+/lo、C-kit+、lin-、CD135-、Slamf1/CD150+；ST-HSC：CD34+、CD38+、SCA-1+、Thy1.1+/lo、C-kit+、lin-、CD135-；MPP：CD34+CD43+CD45+CD38-CD45RA-CD90-CD49f-。
术语“造血多能祖细胞(MPP)”是尚未分化成熟的细胞，造血祖细胞主要由造血干细胞发育分化而来，与造血干细胞相比，它同样具有能够发育分化为全谱系造血类型细胞的潜能，且其同样能够分化为骨髓等；但与造血干细胞的主要区别就是，其不再具有自我更新能力。
术语“造血祖细胞(hematopoietic progenitor cells,HPCs)，也叫专能造血祖细胞”指造血干细胞在一定的微环境和某些因素的调节下，增殖分化为各类血细胞的祖细胞，它也是一种相当原始的具有增殖能力的细胞，但已失去多向分化能力，只能向一个或几个血细胞系定向增殖分化，故也称定向干细胞(committed stem cell)。
术语“重编程”或“去分化”或“增加细胞潜能”或“增加发育潜能”指增加细胞潜能或使细胞去分化为较少分化状态的方法。例如，与非重编程状态中的相同细胞相比较，具有增加的细胞潜能的细胞具有更多发育可塑性(即可分化成更多细胞类型)。换言之，重编程细胞是处于比非重编程状态中的相同细胞较少分化状态的细胞。
术语"分化"是非特异性或较少特异性细胞获得特定细胞(例如血细胞或肌细胞)的特征的过程。分化的或分化诱导性细胞是在细胞谱系中占据更特异性位置的细胞。术语“决定的”应用于分化过程时，是指在已经在分化途径中发展至一个点的细胞，其在正常情况下将继续分化为特定细胞类型或细胞类型亚群，并且在正常情况下不能分化为不同的细胞类 型或者回复到较差分化的细胞类型。
术语“分化标志物基因”或“分化基因”指其表达指示在细胞例如多能细胞内发生的细胞分化的基因。分化标志物基因包括但不限于下述基因：FOXA2、FGF5、SOX17、XIST、NODAL、COL3A1、OTX2、DUSP6、EOMES、NR2F2、NR0B1、CXCR4、CYP2B6、GATA3、GATA4、ERBB4、GATA6、HOXC6、INHA、SMAD6、RORA、NIPBL、TNFSF11、CDH11、ZIC4、GAL、SOX3、PITX2、APOA2、CXCL5、CER1、FOXQ1、MLL5、DPP10、GSC、PCDH10、CTCFL、PCDH20、TSHZ1、MEGF10、MYC、DKK1、BMP2、LEFTY2、HES1、CDX2、GNAS、EGR1、COL3A1、TCF4、HEPH、KDR、TOX、FOXA1、LCK、PCDH7、CD1D FOXG1、LEFTY1、TUJ1、T基因(Brachyury)、ZIC1、GATA1、GATA2、HDAC4、HDAC5、HDAC7、HDAC9、NOTCH1、NOTCH2、NOTCH4、PAX5、RBPJ、RUNX1、STAT1和STAT3。
术语“分化标志物基因谱”或“分化基因谱”、“分化基因表达谱”、“分化基因表达标志”、“分化基因表达组”、或“分化基因标志”指多重分化标志物基因的表达或表达水平。
术语“潜能”指细胞可接近的所有发育选项总和(即发育潜能)。细胞潜能的连续体包括但不限于全能细胞、多能细胞、专能细胞、寡能细胞、单能细胞和终末分化细胞。
术语“多能的”指细胞形成机体或躯体(即胚体)的所有谱系的能力。例如，胚胎干细胞是能够形成来自三个胚层各自的细胞的一类多能干细胞：外胚层、中胚层和内胚层。多能性是发展潜能的连续体，范围从不能产生完整器官的不完全或部分多能性细胞(例如，外胚层干细胞或EpiSC)到能够产生完整器官的原始的、更为多能的细胞(例如，胚胎干细胞)。
术语“诱导多能干细胞”或"诱导性多能干细胞"，或iPSC，意味着干细胞由已诱导或改变的分化的成年/新生儿或胎儿细胞产生，即，重编为能够分化成所有三种胚或真皮层：中胚层，内胚层和外胚层的组织的细胞。所生产的iPSC不是指在自然界中发现的细胞。例如，从血液样品分离成纤维细胞或血细胞，激活，并以逆转录病毒重编程为iPSC。例如，从人血液样品分离成纤维细胞或血细胞，激活，并以逆转录病毒重编程为hiPSC。例如，从人血液样品分离T细胞，激活，并以逆转录病毒重编程为hiPSC。
术语"胚胎干细胞"指胚胎囊胚的内细胞团的天然存在的多能干细胞。胚胎干细胞是多能的，并在发育过程中产生三种主要胚层的所有衍生物：外胚层，内胚层和中胚层。它们对胚胎外膜或胎盘没有贡献，即不是全能的。
术语"专能干细胞"指具有发育潜能以分化成一个或多个胚层(外胚层，中胚层和内胚层)的细胞，但不是全部三种。因此，专能细胞也可以称为“部分分化细胞”。专能细胞是本领域公知的，专能细胞的实例包括成体干细胞，例如造血干细胞和神经干细胞。“专能”表示细胞可能在给定谱系中形成许多类型的细胞，但不能形成其他谱系的细胞。
多能干细胞的分化需要培养系统的变化。最常规的策略是利用拟胚体(EB)的形成作为启动谱系特异性分化的常见和关键中间体。EB是三维团簇，已被证明能模拟胚胎发育，因为它们在其三维区域中产生了许多谱系。
术语“单层分化”是指不同于通过三维多层细胞簇(即“EB形成”)分化的分化方法。单层培养不模拟胚胎发育如EB形成。在一些实例中，将初始hiPSC以单层培养体系接种在维持培养基中并允许扩张直到达到约25％及以上的铺板密度。在一些实例中，将初始hiPSC以单层培养体系接种在维持培养基中并允许扩张直到达到约70％以内的铺板密度。在一些实例中，将初始hiPSC以单层培养体系接种在维持培养基的铺板密度不低于约1500-5000cells/cm2。在一些实例中，将初始hiPSC以单层培养体系接种在维持培养基的铺板密度为约3.7x104-4.7x104cells/cm2及以上。在一些实例中，将初始hiPSC以单层培养体系接种在维持培养基的铺板密度不高于约2.5x105cells/cm2。在一些实例中，将初始hiPSC以单层培养体系接种在维持培养基的铺板密度为约2.5x104-1x105cells/cm2。在具体实施例中，将初始hiPSC以单层培养体系接种在维持培养基的铺板密度为约2.5x104-3x104cells/cm2。在具体实施例中，将初始hiPSC以单层培养体系接种在维持培养基的铺板密度为约8x104-1x105cells/cm2。整体而言，本申请技术方案中涉及的iHSC培养平台、方法均能几乎不依赖于多能干细胞的初始铺板密度的培养条件下，成功诱导获得造血干细胞。
多能性可部分通过评价细胞的多能性特征进行测定。多能性特征包括但不限于：(i)多能干细胞形态；(ii)无限自我更新的潜能，(iii)包括但不限于下述多能干细胞标志物的表达：SSEA1(仅小鼠)、SSEA3/4、SSEA5、TRA1-60/81、TRA1-85、TRA2-54、GCTM-2、TG343、TG30、CD9、CD29、CD133/prominin、CD140a、CD56、CD73、CD90、CD105、OCT4、NANOG、SOX2、CD30和/或CD50；(iv)分化成所有三个体细胞谱系(外胚层、中胚层和内胚层)的能力，(v)由三个体细胞谱系组成的畸胎瘤形成；和(vi)由来自三个体细胞谱系的细胞组成的拟胚体形成。
术语“多能干细胞形态”指胚胎干细胞的典型形态特点。正常胚胎干细胞形态的特征在于圆且小的形状、具有高核质比、核仁的显著存在和典型细胞间间隔。
术语“贴壁”指在适当培养基的存在下，细胞附着至容器，例如细胞附着至无菌塑料(或涂布塑料)细胞培养皿或烧瓶。某些类别的细胞在培养中无法持续或不生长，除非它们贴壁至细胞培养容器。某些类别的细胞(“非贴壁细胞”)无需贴壁在培养中维持和/或增殖。
术语“细胞外基质(ECM)或基质”，指多细胞有机体中，细胞周围由多种大分子组成的复杂网络所还有的物质及对应形成的结构。细胞外基质主要由5类物质组成，即胶原蛋白、非胶原蛋白、弹性蛋白、蛋白聚糖与氨基聚糖，按分布部位划分主要分为基底膜和间质基质。在细胞培养中使用的细胞外基质多为提取物或化学合成或生物表达提纯的组成即形成结构相对稳定的细胞外支持物质。
术语“培养”或“细胞培养”指在体外环境中的细胞维持、生长和/或分化。“细胞培养基”、“培养基”、“补充物”和“培养基补充物”指用于培养细胞的营养组合物。在本申请的一些方面，所述的一个或多个分化阶段可在无滋养层、无血清条件下进行。在一些实例中，iPSC可接种在维持培养基中。在一些实例中，维持培养基包括Stem Cell Technologies(Vancouver,Canada)的mTeSRTM1或TeSRTM1plus，Stem Cell  Technologies(Vancouver,Canada)的MyeloCultTMH5100或StemSpanTMSFEM II,Gibco的E8medium，Gibco的StemProTM-34SFM(1X)。
本申请提供的培养平台的一种或多种培养基是无滋养层的环境。在其它实施方式中，细胞培养基含有过多的补充物，例如血清、提取物、生长因子、激素、细胞因子等。通常，培养平台包含一种或多种阶段特异性无滋养层、无血清培养基，每种培养基还包含以下一种或多种：合适的营养/提取物细胞因子和培养基添加剂。合适的营养/提取物可包括例如DMEM/F-12(Dulbecco'sModified Eagle Medium/Nutrient Mixture F-12)，其是广泛使用的用于支持许多不同哺乳动物细胞生长的基础培养基；Neurobasal medium在人的多种细胞培养中被广泛用作基础培养基；L-glutamin。其他培养基添加剂可以包括但不限于N2、B27、β-Me、抗氧化剂(例如抗坏血酸)。在一些实例中，本申请的诱导体系包含一种或多种以下细胞因子或小分子化合物：CHIR99021、SB4、VEGF、Forskolin、Deoxyshikonin、Isoarnebin I、Isoarnebin 4、Resveratrol、VPA、SB431542、E-616452、A83-01、UM729、UM171、SR1，这些细胞因子或小分子化合物可以商购获得，例如购自R&D Systems(Minneapolis，Minn.)、Selleck公司等，也可以是天然的/重组的或化学合成。在一些实例中，本申请的培养体系包含一种或多种小分子化合物:GSK3β抑制剂(例如CHIR99021、SB216763、CHIR-98014、LY2090314、BIO-acetoxime、Alsterpaullone、CP21R7)、VEGF通路激活剂(例如arnebin、Deoxyshikonin、Isoarnebin I、Isoarnebin 4)、cAMP通路激活剂(例如Forskolin)、Notch信号通路激活剂(例如Resveratrol、VPA)、TGFβ受体/ALK抑制剂(例如SB431542、E-616452、A83-01)、造血干细胞促增殖分子(例如UM729、UM171、SR1)、HIF1a激活剂(例如Roxadustat、IOX2、DMOG、PT-2385、Belzutifan、PX-478、LW6、Oltipraz、Daprodustat、BAY87-2243、KC7F2、Molidustat、PT2399、Amifostine、Enarodustat、Chlorogenic acid、ML228、Acriflavine、Vadadustat、FG-2216、Tilorone dihydrochloride、Fraxinellone、1,4-DPCA、MK-8617、Glucosamine hydrochloride、Hydralazine hydrochloride、AKBA)。在一些实例中，细胞因子/小分子化合物在浓度方面是根据经验确定或由已建立的细胞因子/小分子化合物技术指导的阶段和/或细胞类型特异性的。
术语“维持”指在组织或机体外，例如在无菌塑料(或涂布塑料)细胞培养皿或烧瓶中的细胞持续、繁殖(生长)和/或分化。“培养”或“维持”可利用培养基作为营养素、激素和/或帮助使细胞繁殖和/或持续的其他因子的源。
在本申请的各个培养方法、培养平台中使用iHSC-A培养基时，所述iHSC-A包含SB4和CHIR99021。
在一个实施方式中，在使用的各培养基中化合物A的浓度范围为0.1-20μM。示例的，培养基中化合物A浓度范围是0.1-20μM，优选地，为0.1-15μM，更优选地，为0.1-10μM、具体实施例中，在使用的各培养基中SB4浓度范围是0.1-20μM。示例的，培养基中SB4浓度范围是0.1-20μM，优选地，为0.1-15μM，更优选地，为0.1-10μM，例如为0.2μM、0.3μM、 0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
具体实施例中，在使用的各培养基中CHIR99021浓度范围是0.1-20μM。示例的，培养基中SB4浓度范围是0.1-20μM，优选地，为0.1-15μM，更优选地，为0.1-10μM，例如为0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
每个步骤中所述细胞群的分化程度可通过监测和/或检测分化细胞群中一个或多个细胞标记物的表达来确定。例如，可以测定具有更高分化细胞类型特征的标记物表达的增加或具有较低分化细胞类型特征的标记物表达的减少。细胞标记物的表达可以通过任何合适的技术来确定，包括：免疫细胞化学、免疫荧光、RT-PCR、免疫印迹、荧光激活细胞分选(fluorescence activated cell sorting,FACS)和酶分析。例如，本文所述不表达标记物的细胞可表现标记基因的活跃转录和细胞内表达，但所述细胞的表面可不存在可检测水平的所述标记物。
通过本文所述方法中的步骤产生的部分分化细胞群，例如为非功能性T细胞，如iPSC、中胚层、造血内皮、HSC、MPP、HPC、T祖细胞，可在下一个分化步骤之前进行培养、维持或扩增。部分分化的细胞可通过任何方便的技术进行扩增。
在每个步骤之后，在所述培养基中培养后，所述部分分化细胞群可包含1％或更多、5％或更多、10％或更多、15％或更多的部分分化细胞。如果需要，部分分化细胞群可通过任何方便的技术(如MACs或FACS)进行纯化。
本申请提供了用于在单层培养中从多能干细胞分化和扩增造血细胞谱系的方法和组合物，其包括使多能性干细胞与包括小分子SB4和CHIR99021的诱导组合物I接触，获得并扩增了造血相关的中胚层细胞，而不形成拟胚体。本申请提供了用于在单层培养中从多能干细胞分化和扩增造血细胞谱系的方法和组合物，其包括使多能性干细胞来源的中胚层细胞与包括小分子Deoxyshikonin、Isoarnebin I和/或Isoarnebin 4，和Forskolin组成的诱导组合物II接触，获得并扩增了造血内皮，而不形成拟胚体。本申请提供了用于在单层培养中从 多能干细胞分化和扩增造血细胞谱系的方法和组合物，其包括使多能性干细胞来源的造血内皮与包括小分子Resveratrol和SB431542，可选的UM729组成的诱导组合物III接触，获得并扩增了造血干细胞，而不形成拟胚体。本申请提供了用于在单层培养中从多能干细胞分化和扩增造血细胞谱系的方法和组合物，其包括使多能性干细胞来源的造血相关的中胚层细胞与包括小分子Resveratrol和SB431542，可选的Forskolin、可选的Deoxyshikonin、Isoarnebin I和/或Isoarnebin 4组成的诱导组合物V接触，获得并扩增了的造血干细胞，而不形成拟胚体。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含VEGF和Deoxyshikonin。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含VEGF和Isoarnebin I。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含VEGF、Deoxyshikonin和Forskolin。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含Deoxyshikonin和Forskolin。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含VEGF和Forskolin。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含VEGF、Isoarnebin I和Forskolin。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含Isoarnebin I和Forskolin。
在本申请的各个培养方法、培养平台中使用iHSC-B培养基时，在一个具体的实施方式中，所述iHSC-B包含Isoarnebin 4和Forskolin。
在本申请的各个培养方法、培养平台中使用iHSC-C培养基时，在一个具体的实施方式中，所述iHSC-C包含Resveratrol和SB431542。
在本申请的各个培养方法、培养平台中使用iHSC-C培养基时，在一个具体的实施方式中，所述iHSC-C包含Resveratrol、SB431542和UM729。
在本申请的各个培养方法、培养平台中使用iHSC-D培养基时，在一个具体的实施方式中，所述iHSC-D包含Forskolin、Resveratrol、SB431542、VEGF和Deoxyshikonin。
在本申请的各个培养方法、培养平台中使用iHSC-D培养基时，在一个具体的实施方式中，所述iHSC-D包含Forskolin、Resveratrol、SB431542、和Deoxyshikonin。
在本申请的各个培养方法、培养平台中使用iHSC-D培养基时，在一个具体的实施方式中，所述iHSC-D包含Forskolin、Resveratrol、SB431542、和VEGF。
在本申请的各个培养方法、培养平台中使用iHSC-D培养基时，在一个具体的实施方式中，所述iHSC-D包含Forskolin、Resveratrol、SB431542、和Isoarnebin I。
在本申请的各个培养方法、培养平台中使用iHSC-D培养基时，在一个具体的实施方式中，所述iHSC-D包含Forskolin、Resveratrol、SB431542、和Isoarnebin 4。
在本申请的各个培养方法、培养平台中使用iHSC-D培养基时，在一个具体的实施方式中，所述iHSC-D包含Forskolin、Resveratrol、SB431542、VEGF和Isoarnebin I。
例如，iHSC-B或iHSC-D，包括1-10μM Deoxyshikonin；优选地，1-6μM Deoxyshikonin，更优选地，2-6μM Deoxyshikonin，或包括1-20μM Isoarnebin I，优选地，5-15μM Isoarnebin I，更优选地，8-15μM Isoarnebin I，或包括1-20μM Isoarnebin 4，优选地，5-15μM Isoarnebin4，更优选地，8-15μM Isoarnebin 4；还包括1-20μM Forskolin；优选地，2-10μM Forskolin更优选地，2-4μM Forskolin；还包括StemPro34。
具体实施例中，在使用的各培养基中Deoxyshikonin浓度范围是1-20μM。示例的，培养基中Deoxyshikonin浓度范围是2-15μM，优选地，为3-18μM，例如可以为1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
具体实施例中，在使用的各培养基中Isoarnebin I浓度范围是1-20μM。示例的，培养基中Isoarnebin浓度范围是2-15μM，优选地，为3-18μM，例如可以为1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
具体实施例中，在使用的各培养基中Isoarnebin 4浓度范围是1-20μM。示例的，培养基中Isoarnebin浓度范围是2-15μM，优选地，为3-18μM，例如可以为1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
具体实施例中，在使用的各培养基中Forskolin浓度范围是0.2-10μM。示例的，培养基中Forskolin浓度范围是0.2-10μM，优选地，为0.5-8μM，更优选地，为1-5μM，例如为0.2μM、 0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM。
具体实施例中，在使用的各培养基中Resveratrol浓度范围是2-20μM。示例的，培养基中Resveratrol浓度范围是3-18μM，优选地，为5-15μM，例如可以为2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
具体实施例中，在使用的各培养基中SB431542浓度范围是2-20μM。示例的，培养基中SB431542浓度范围是3-18μM，优选地，为5-15μM，例如可以为2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM、5.1μM、5.2μM、5.3μM、5.4μM、5.5μM、5.6μM、5.7μM、5.8μM、5.9μM、6.0μM、6.5μM、7.0μM、7.5μM、8.0μM、8.5μM、9.0μM、9.5μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM。
具体实施例中，在使用的各培养基中UM729浓度范围是0.1-5μM。示例的，培养基中UM729浓度范围是0.3-4μM，优选地，为0.5-4.5μM，例如为0.1μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.1μM、2.2μM、2.3μM、2.4μM、2.5μM、2.6μM、2.7μM、2.8μM、2.9μM、3.0μM、3.1μM、3.2μM、3.3μM、3.4μM、3.5μM、3.6μM、3.7μM、3.8μM、3.9μM、4.0μM、4.1μM、4.2μM、4.3μM、4.4μM、4.5μM、4.6μM、4.7μM、4.8μM、4.9μM、5.0μM。
本文提供的利于获得造血相关的中胚层、造血内皮、造血干细胞的单层培养平台，导致造血干细胞和分化后代如T、B、NKT和NK细胞的衍生。所示单层分化策略将增强的分化效率与大规模扩增结合起来，可以递送治疗相关数量的多能干细胞来源的造血细胞谱系用于各种治疗应用。此外，本申请公开了使用本文提供的方法的单层培养产生造血细胞谱系，其能够进行全范围的体外分化，离体调节和体内长期造血自我更新、重建和移植。本申请提供的包括一种或多种选自小分子的诱导组合物是一种安全、高效、低成本的造血细胞谱系分化方法。
本申请涉及初始多能干细胞向非多能细胞或部分分化细胞分化的多阶段过程，包括， 造血相关的中胚层细胞，造血内皮，造血干细胞、造血祖细胞、造血多能祖细胞(MPP)、T祖细胞、NK祖细胞；或全分化的末端造血细胞，例如，如，T细胞、B细胞、NKT细胞或NK细胞。本申请还涉及所公开的方法中使用的组合物；以及由所公开的方法产生的细胞群、细胞系或克隆细胞。
通过在包含GSK3β抑制剂和化合物SB4的培养基中接种克隆iPSC细胞来获得源自iPSC的造血细胞谱系，并使用分步进行的策略在分化初期和中期应用小分子诱导剂组合。因此，本申请能够将扩增的克隆iPSC直接转移到单层形式的贴壁培养中，以便立即分化，而不需要从iPSC形成EB。
本申请提供了在无血清/无滋养层条件下，且在可扩展和单层培养平台中，不需要EB形成的情况下，添加包括小分子SB4和CHIR99021的诱导组合物能诱导多能干细胞(包括hiPSC)分化为造血相关的中胚层细胞。本申请所述诱导组合物也可以在EB形成的情况下，诱导多能干细胞(包括hiPSC)分化为中胚层细胞。示例性，所述中胚层为有利于分化造血细胞谱系的稳定高效的中胚层细胞。具体实施例中，包括进一步诱导分化所述中胚层细胞生成造血内皮、造血干细胞、MPP，产生造血细胞谱系的方法和组合物。
在一些实例中，上述iPSC分化的一个或多个阶段可以在无滋养层条件下进行。这种无滋养层条件可以是包括但不限于单层培养和悬浮培养的形式。在一些实例中，多能干细胞向中胚层细胞的分化是在单层无滋养层条件下进行的。在一些实例中，在单层无滋养层条件下进行中胚层细胞向造血内皮的分化。在一些实例中，造血内皮向造血干细胞的分化是在单层无滋养层条件下进行的。在一些实例中，造血干细胞向T祖细胞或NK祖细胞的分化是在悬浮无滋养层条件下进行的，或者先在单层无滋养层条件下进行，随后在悬浮无滋养层条件下进行。在一些实例中，T祖细胞分化为全分化T细胞或NK祖细胞分化为全分化NK细胞是在悬浮无滋养层条件下进行的，或者先在单层无滋养层条件下进行，随后在悬浮无滋养层条件下进行。
在本文考虑的组合物和细胞培养基中的小分子的量可改变，并且可根据具体培养条件进行优化，包括所使用的具体分子和组合、在培养基中培养的细胞类型以及具体应用。
在一些实例中，小分子存在于组合物中的浓度足以诱导造血内皮及进一步的造血分化，最终实现高效稳定的造血细胞谱系分化。
本申请提供了使用无滋养层、无血清条件、不含细胞因子的培养基的培养平台，其支持iPSC在单层培养中直接分化而不需要来自iPSC的EB或聚集中间体。
培养平台
用于培养多能干细胞的现有方法主要依赖于滋养层细胞或用滋养层细胞预条件化并含有胎牛血清的培养基；然而，这种环境可能不适用于生产用于临床和治疗用途的细胞。本文考虑的无滋养层层环境，有助于制造临床级细胞系，特别是hESC、hiPSC和多能干细胞来源的HSC、MPP、T、B、NKT或NK细胞系。
在一些实例中，无滋养层环境基本上不含人滋养层细胞，并且不由滋养层细胞预条件 化，包括但不限于小鼠胚胎成纤维细胞、人成纤维细胞、角质形成细胞和胚胎干细胞。无滋养层细胞培养基适用于培养多能细胞、重编程细胞、单细胞培养、多能细胞的解离和传代、多能细胞的分选、基态多能细胞的产生、基态多能性的维持和/或诱导多能细胞分化。在一些实例中，无滋养层环境用于诱导多能性、改善重编程的效率、增加或维持细胞的潜能和/或诱导分化。在一些实例中，无滋养层条件不含细胞因子和生长因子，包括bFGF。
在一些实例中，上述iPSC分化的一个或多个阶段可以在无滋养层条件下进行。这种无饲料条件包括但不限于单层培养和悬浮培养的形式。在一些实例中，多能干细胞向造血相关的中胚层细胞的分化是在单层无滋养层条件下进行的。在一些实例中，在单层无滋养层条件下进行中胚层细胞向造血内皮的分化。在一些实例中，造血内皮向造血干细胞、MPP的分化是在单层无滋养层条件下进行的。在一些实例中，造血干细胞向MPP、造血祖细胞、T祖细胞或NK祖细胞的分化是在悬浮无滋养层条件下进行的，或者先在单层无滋养层条件下进行，随后在悬浮无滋养层条件下进行。在一些实例中，T祖细胞分化为全分化T细胞或NK祖细胞分化为全分化NK细胞是在悬浮无滋养层条件下进行的，或者先在单层无滋养层条件下进行，随后在悬浮无滋养层条件下进行。
任何合适的培养瓶或细胞培养容器可用作添加基本培养基和/或附加培养基的细胞培养物的载体。在一些实例中，用粘附促进基质/底物(例如，胶原，纤连蛋白，含RGD的多肽，明胶等)涂覆培养容器的表面会促进细胞的附着，并且在具体实施方式中可以增强本文公开的细胞基本培养基和附加培养基的作用。用于培养和传代细胞的合适底物是本领域已知的，并且包括但不限于玻连蛋白(Vitronectin,VTN)、明胶、层粘连蛋白(Laminin，LN)、纤连蛋白(Fibronectin，FN)、胶原IV(Collagen IV)、弹性蛋白、骨桥蛋白、血小板反应素、天然存在的细胞系产生的基质如MatrigelTM的混合物和合成或人造表面如聚胺单层和羧基封端的单层。在一些实例中，提供无滋养层条件包括在基质涂覆的表面上培养细胞。在一些实例中，本文考虑的培养平台包括含有matrigel、VTN-N、laminin-511和/或laminin-521基质/底物。
在一些实例中，上述一个或多个分化阶段可在无血清条件下进行。适于细胞贴壁和/或诱导的可商购的无血清培养基的例子包括S t e m C e l l T e c h n o l og i e s(Vancouver,Canada)的mTeSRTM1，mTeSRTM2，mTeSR plus medium，TeSRTM-AOF medium,StemSpanTM SFEM II，StemSpan CD34+Expansion Supplement(10x)，StemSpanTM-AOF，ReproCELL(Boston,MA)的Primate ES/iPS细胞培养基，Invitrogen(Carlsbad,CA)的StemProTM-34SFM(1X)，Essential 8TM培养基，CST Essential 8TM培养基，和味之素的StemFit basic03。
在一些些实例中，培养平台的一种或多种培养基是无滋养层的环境，并且任选地基本上不含细胞因子和/或生长因子。在其它实施方式中，细胞培养基含有补充物，例如血清、提取物、生长因子、激素、细胞因子等。通常，培养平台包含一种或多种阶段特异性无滋养层、无血清培养基，每种培养基还包含以下一种或多种：营养物/提取物、生长因子、 激素、细胞因子和培养基添加剂。合适的营养/提取物可包括例如DMEM/F-12(Dulbecco'sModified Eagle Medium/Nutrient Mixture F-12)，其是广泛使用的用于支持许多不同哺乳动物细胞生长的基础培养基；KOSR(剔除血清替代)；L-glut；NEAA(非必需氨基酸)。其他培养基添加剂可以包括但不限于MTG、ITS、βME、抗氧化剂(例如抗坏血酸)。在一些些实例中，本申请的培养基包含一种或多种以下细胞因子或生长因子：表皮生长因子(EGF)、酸性成纤维细胞生长因子(aFGF)、碱性成纤维细胞生长因子(bFGF)、白血病抑制因子(LIF)、肝细胞生长因子(HGF)、胰岛素样生长因子1(IGF-1)、胰岛素样生长因子2(IGF-2)、角化细胞生长因子(KGF)、神经生长因子(NGF)、血小板源生长因子(PDGF)、转化生长因子β(TGF-β)、骨形态发生蛋白(BMP4)、血管内皮细胞生长因子(VEGF)转铁蛋白、各种白细胞介素(如IL-1至IL-18)、各种集落刺激因子(如粒细胞/巨噬细胞集落刺激因子(GM-CSF))、各种干扰素(如IFN-γ)和对干细胞有影响的其他细胞因子，如干细胞因子(SCF)和促红细胞生成素(EPO)。这些细胞因子可以商购获得，例如购自R&D Systems(Minneapolis，Minn.)，也可以是天然的或重组的。在一些实例中，本申请的培养基包含一种或多种骨形态发生蛋白(BMP4)、胰岛素样生长因子-1(IGF-1)、碱性成纤维细胞生长因子(bFGF)、血管内皮生长因子(VEGF)、造血生长因子(例如SCF、GMCSF、GCSF、EPO、IL3、TPO、EPO)、Fms相关酪氨酸激酶3配体(Flt3L)；和来自白血病抑制因子(LIF)、IL3、IL6、IL7、IL11、IL15的一种或多种细胞因子。在一些实例中，生长因子/有丝分裂原和细胞因子在浓度方面是根据经验确定或由已建立的细胞因子技术指导的阶段和/或细胞类型特异性的。
通常，用于分化诱导性多能细胞的技术涉及使用基于多核苷酸、多肽和/或小分子的方法直接或间接调节特定细胞途径。例如通过使细胞与一种或多种调节剂接触来调节细胞的发育潜能。本文所用的“接触”包括在一种或多种因素(例如小分子、蛋白质、肽等)的存在下培养细胞。在一些实例中，将细胞与一种或多种试剂接触以诱导细胞分化。这种接触可以通过在体外培养期间将一种或多种试剂引入细胞来进行。因此，可以通过将一种或多种试剂引入细胞培养基中的细胞来进行接触。细胞可以保持在包含一种或多种试剂的培养基中足以使细胞达到所需分化表型的时间。在一些实例中，当通过载体将一个或多个因子引入细胞时，发生“接触”。在一实例中，一种或多种载体由逆转录病毒、仙台病毒、腺病毒、游离体、微环、具有表达盒的载体系统或mRNA引入。
在其它实施方式中，如本文所公开的培养平台的一种或多种阶段特异性的无滋养层、无血清培养基包含一种或多种小分子。在一些实例中，培养平台包括含有GSK3β抑制剂、Wnt通路激活剂、VEGF通路激活剂、Notch信号通路激活剂、TGFβ受体/ALK抑制剂的细胞培养基，并且不包含MEK抑制剂。
本文考虑的培养平台还通过利用具有降低的自发分化和/或达到基础状态多能性的工业或临床级多能细胞的均匀群体来提供许多优点。在一些实例中，均质iPSC保持在包含ROCK抑制剂维持培养基中。如本文使用的，术语“均质”是指细胞群，其中每个细胞与群 体中的其它细胞相同或基本相同。在一些实例中，如果每个细胞表达一种或多种与本文所考虑的相同的多能性标记，例如SSEA4、TRA-1-81、TRA-1-60，则细胞与群体中的其它细胞相同。在一些实例中，如果至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更多的细胞相同或基本上是与群体中的其他细胞相同，则群体是均质的。
在多个实施方式中，用于通过本申请的造血内皮产生造血细胞谱系的培养平台的细胞培养基包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，不含或基本上不含GSK3β抑制剂、Wnt通路激活剂、MEK抑制剂和ROCK抑制剂、cAMP通路激活剂和/或VEGF通路激活剂。在多个实施方式中，用于通过本申请的中胚层细胞产生造血细胞谱系的培养平台的细胞培养基包含cAMP通路激活剂、VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，不含或基本上不含GSK3β抑制剂、Wnt通路激活剂、MEK抑制剂和ROCK抑制剂。在多个实施方式中，用于通过本申请的iPSC细胞产生造血细胞谱系的培养平台的细胞培养基包含化合物A、GSK3β抑制剂、Wnt通路激活剂、cAMP通路激活剂、VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，不含或基本上不含MEK抑制剂。在一些实例中，培养平台包括维持培养基，用于保持初始hiPSC的接种培养基，该培养基包含ROCH抑制剂。本申请人不希望受任何具体理论的约束，发现小分子SB4与CHIR99021组成的分化诱导剂可以在不存在BMP4、Activin A的情况下，实现高效的造血方向分化，且几乎不受细胞铺板密度影响。已有研究发现高浓度的VEGF有利于促进动脉内皮的定向分化，而在本申请的中胚层向造血分化过程中发明人意外地发现，高浓度的VEGF，尤其在结合cAMP激活剂时；或VEGF通路激活剂，尤其在结合cAMP激活剂时，均能诱导获得动脉和造血两种类型为主的内皮群体，其中动脉内皮的存在大大促进了造血内皮的造血分化。
如本文所使用的，术语“长期”基于但不限于传代的数量来测量，通常意味着至少10、15、20、25、30、35、40、45、50或更多代数。如所界定的那样，“传代”是指当细胞增殖到期望的程度时，将细胞分开并铺展到多细胞培养表面或培养瓶中的行为。在一些实例中，均质iPSC保持在包含ROCK抑制剂的维持培养基中。
1.化合物A
本申请中术语“化合物A”的结构如式I所示：
其中，R1选自：
X1选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素；X2选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素；X3选自H、甲基、乙基、羟基、甲氧基、乙氧基、卤素；其 中，R2为：
所述X4、X5、X6、或X7选自H、甲基、乙基、羟基、甲氧基、乙氧基、或卤素，X4、X5、X6、或X7可以相同，也可以不同；
优选的，所述R2选自以下任一结构：
本申请中术语“SB4”的结构如式II所示：
2.Wnt通路激活剂
本申请中术语“Wnt通路激活剂”指Wnt信号传导途径的激活剂，包括但不限于Wntl、Wnt2、Wnt2b/13、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt7c、Wnt8、Wnt8a、Wnt8b、Wnt8c、Wnt10a、Wntl0b、Wnt11、Wnt14、Wnt15和Wnt16中的一种或多种的激活剂。
3.GSK3β抑制剂
本申请中术语“GSK3β抑制剂”具有激活Wnt途径作用，包括但不限于多核苷酸、多肽和小分子。GSK3β抑制剂可减少GSK3β表达和/或GSK3β活性。GSK3β抑制剂包括但不限于抗GSK3β抗体、显性失活GSK3β变体、靶向GSK3β的siRNA或shRNA或miRNA或反义核酸。在一些实例中，GSK3β抑制剂包括但不限于：肯帕罗酮、1-氮杂坎帕罗酮、CHIR99021、CHIR98014、AR-A014418、CT 99021、CT 20026、SB216763、AR-A014418、锂、SB415286、TDZD-8、BIO、BIO-丙酮肟、(5-甲基-1H-吡唑-3-基)-(2-苯基喹唑啉-4-基)胺、吡啶卡巴唑-环戊二烯基钌络合物、TDZD-8 4-苄基-2-甲基-1,2,4-噻二唑烷-3-,5-二酮、2-硫代(3-碘苄基)-5-(1-吡啶基)-[1,3,4]-噁二唑、OTDZT、α-4-二溴苯乙酮、AR-AO 144-18、3-(1-(3-羟丙基)-1H-吡咯并[2,3-b]吡啶-3-基]-4-吡嗪-2-基-吡咯-2,5-二酮；TWSl 19吡咯并嘧啶化合物、L803H-KEAPPAPPQSpP-NH2或其肉豆蔻酰化形式；2-氯-1-(4,5-二溴-噻吩-2-基)-乙酮； GF109203X；RO318220；TDZD-8；TIBPO；和OTDZT。在一实例中，GSK3β抑制剂是CHIR99021、BIO或肯帕罗酮。在一些实例中，GSK3β抑制剂是CHIR99021。在一些实例中，GSK3β抑制剂是BRD0705。在一些实例中，CHIR99021浓度范围是0.1-20μM，优选地，为0.1-15μM，更优选地，为0.1-10μM。
4.VEGF(血管内皮生长因子)通路激活剂
VEGF通路激活剂包括下述中的一种或多种：包括但不限于多核苷酸、多肽和小分子化合物。包括但不限于VEGF121、VEGF145、VEGF162、VEGF165、VEGF183、VEGF189和VEGF206的一种或多种的激活剂。包含编码VEGF多肽的核苷酸序列的核酸、包含VEGF多肽的氨基酸序列的多肽、包含编码活化VEGF受体的核苷酸序列的核酸、包含活化VEGF受体的氨基酸序列的多肽、促进VEGF信号传导的小有机分子、抑制VEGF拮抗剂的表达或活性的小有机分子、抑制VEGF拮抗剂表达的反义寡核苷酸、抑制VEGF拮抗剂表达的核酶、抑制VEGF拮抗剂表达的RNAi构建体或siRNA或shRNA、与VEGF拮抗剂结合且抑制VEGF拮抗剂活性的抗体、包含编码VEGF蛋白多肽的核苷酸序列的核酸、包含VEGF蛋白多肽的氨基酸序列的多肽、包含编码VEGF受体的核苷酸序列的核酸，包含VEGF受体的氨基酸序列的多肽。在一些实例中，VEGF通路激活剂包括Isoarnebin I、Isoarnebin 4、Deoxyshikonin或其组合。
术语“Isoarnebin I”，别称β,β-Dimethylacrylshikonin(Isoarnebin I)，是从Arnebia nobilis中提取得到的一种萘醌衍生物。Isoarnebin I的结构如下所示：
术语“Deoxyshikonin”，从紫草Lithospermum erythrorhizon中分离，能增加HMVEC-dLy中VEGF-C和VEGF-A mRNA的表达。Deoxyshikonin的结构如下所示：
术语“Isoarnebin 4”，也称Shikonin，是一种Pyruvate kinase M2(PKM2)有效的、特异的抑制剂。Isoarnebin4的结构如下所示：
5.cAMP通路激活剂
cAMP通路激活剂包括下述中的一种或多种：包含编码cAMP多肽的核苷酸序列的核酸、包含cAMP多肽的氨基酸序列的多肽、包含编码活化cAMP受体的核苷酸序列的核酸、包含活化cAMP受体的氨基酸序列的多肽、促进cAMP信号传导的小有机分子、抑制cAMP拮抗剂的表达或活性的小有机分子、抑制cAMP拮抗剂表达的反义寡核苷酸、抑制cAMP拮抗剂表达的核酶、抑制cAMP拮抗剂表达的RNAi构建体或siRNA或shRNA、与cAMP拮抗剂结合且抑制cAMP拮抗剂活性的抗体、包含编码cAMP蛋白多肽的核苷酸序列的核酸、包含cAMP蛋白多肽的氨基酸序列的多肽、包含编码cAMP受体的核苷酸序列的核酸，包含cAMP受体的氨基酸序列的多肽。在一些实例中，cAMP通路激活剂包括Forskolin和/或PACAP 1-38。
术语“Forskolin”,也称Colforsin是一种腺苷酸环化酶激活剂，分子式为C22H34O7，结构如下所示：
术语“PACAP 1-38”，即Pituitary Adenylate Cyclase Activating Polypeptide 38，是高度有效的PACAP受体激动剂，能够刺激腺苷酸环化酶。PACAP 1-38的结构如下所示：
6.Notch信号通路激活剂
Notch包括Notch受体家族的所有成员，包括但不限于Notch1。Notch信号通路激活剂包括但不限于Notch受体的激动剂。Notch信号通路激动剂将结合Notch受体，并且还引发或介导与Notch受体相关的信号传导事件，例如引起Notch的细胞内结构域被切割并易位于细胞核。
Notch信号通路激活剂包括下述中的一种或多种：包含编码Notch多肽的核苷酸序列的核酸、包含Notch多肽的氨基酸序列的多肽、包含编码活化Notch受体的核苷酸序列的核酸、包含活化Notch受体的氨基酸序列的多肽、促进Notch信号传导的小有机分子、抑制Notch拮抗剂的表达或活性的小有机分子、抑制Notch拮抗剂表达的反义寡核苷酸、抑制Notch拮抗剂表达的核酶、抑制Notch拮抗剂表达的RNAi构建体或siRNA或shRNA、与Notch拮抗剂结合且抑制Notch拮抗剂活性的抗体、包含编码Notch蛋白多肽的核苷酸序列的核酸、包含VEGF蛋白多肽的氨基酸序列的多肽、包含编码Notch受体的核苷酸序列的核酸，包含Notch受体的氨基酸序列的多肽。Notch信号通路激活剂包括但不限于Jag1、Jag2、DLL-1、 DLL-3和DLL-4。Notch信号通路激活剂包括但不限于EP 2606884、US 6689744和US5780300中公开的那些，其公开内容通过引用并入本文。在一些实例中，一种或多种Notch配体可以作为可溶性肽引入，或固定在固体材料上。固体材料可以包括但不限于聚苯乙烯板或珠。用于Notch配体固定化的珠可以是琼脂糖珠、磁珠和乳胶珠。在一些实例中，Notch配体肽与珠缀合/固定。在另一个实施方式中，Notch配体肽与聚苯乙烯板的表面缀合/固定。在一些实例中，Notch配体的固定是非共价的。在一些实例中，Notch配体肽由细胞呈递。在一些实例中，Notch信号通路激活剂包括Resveratrol和/或VPA。
术语“Resveratrol”也称为SRT501或trans-Resveratrol,具有广泛靶点，包括环氧酶、脂肪氧合酶、sirtuins和其他蛋白质。Resveratrol的结构如下所示：
术语“VPA”，即Valproic acid(VPA,2-Propylvaleric Acid,Valproate)，是一种histone deacetylase(HDAC)抑制剂，可激活Notch-1信号。VPA的结构如下所示：
7.TGFβ受体/ALK抑制剂
术语“TGFβ受体/ALK抑制剂”,即包括的TGFβ受体抑制剂通过抑制剂ALK通路起作用。包括针对TGFβ受体(例如ALK5)的抗体、TGFβ受体的显性失活变体、抑制TGFβ受体表达的反义核酸、或抑制TGFβ受体/ALK信号通路的小分子化合物。示例性的TGFβ受体/ALK抑制剂包括但不限于SB431542；A-83-01，也称为3-(6-甲基-2-吡啶)-N-苯基-4-(4-喹啉)-1H-吡唑-1-硫代酰胺；RepSox(即E616452)；2-(3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-1,5-萘啶；Wnt3a/BIO；GW788388(-{4-[3-(吡啶-2-基)-1H-吡唑-4-基]吡啶-2-基}-N-(四氢-2H-吡喃-4-基)苯甲酰胺)；SM16(参见，例如，Suzuki等，Cancer Research67(5):2351-2359(2007))；IN-1130(3-((5-(6-甲基吡啶-2-基)-4-(喹喔啉-6-基)-1H-咪唑-2-基)甲基)苯甲酰胺)；GW6604(2-苯基-4-(3-吡啶-2-基-1H-吡唑-4-基)吡啶)；SB-505124(2-(5-苯并[1,3]二氧杂环戊烯-5-基-2-叔丁基-3H-咪唑-4-基)-6-甲基吡啶盐酸盐)；以及嘧啶衍生物(参见，例如，列于Stiefl等，WO2008/006583中的那些，在此引入作为参考)。示例性TGFβ受体/ALK抑制剂，包括但不限于：TGFβ受体抑制剂、SMAD 2/3磷酸化抑制剂、SMAD 2/3和SMAD 4相互作用抑制剂、以及SMAD 6和SMAD 7激活物/激动剂。在一些实例中，TGFβ受体抑制剂的具体例子包括但不限于SU5416；2-(5-苯并[1,3]二氧杂环戊烯-5-基-2-叔丁基-3H-咪唑-4-基)-6-甲基吡啶盐酸盐(SB-505124)；lerdelimumb(CAT-152)；美替木单抗(CAT-192)；GC-1008；ID11；AP-12009；AP-11014；LY550410；LY580276；LY364947； LY2109761；SB-505124；SB-431542；SD-208；SM16；NPC-30345；Ki26894；SB-203580；SD-093；Gleevec；3,5,7,2',4'-五羟基黄酮(Morin)；激活素-M108A；P144；可溶性TBR2-Fc。
在一些实例中，TGFβ受体/ALK抑制剂包括SB431542、A-83-01、E-616452或其组合。
术语“SB431542”，是TGFβ受体/ALK5抑制剂,结构如下所示：
术语“A-83-01”，是TGF-βtype I receptor(ALK5-TD)的抑制剂，还抑制由ALK4-TD和ALK7-TD诱导的转录。A-83-01的结构如下所示：
术语“E-616452”，又称RepSox、SJN 2511、ALK5 Inhibitor II，是一种有效的选择性TGFβR-1/ALK5抑制剂。E-616452的结构如下所示：
8.造血干细胞促增殖分子
造血干细胞促增殖分子包括SCF、FL、IL-1、IL-12等细胞因子、或小分子化合物。在一些实例中，造血干细胞促增殖分子包括UM729、UM171、SR1或其组合。
术语“UM729”，是一种嘧啶-[4,5-b]-吲哚衍生物，可在体外增强人类造血干细胞的自我更新。UM729的结构如下所示：
术语“UM171”，是人类造血干细胞自我更新的有效激动剂，独立于AhR抑制。UM171
术语“SR1”，即StemRegenin 1。SR1的结构如下所示：
本申请提供了分选或富集的多能干细胞来源的HSC、MPP细胞沿着造血谱系进一步分化为各种特定细胞类型，包括T细胞和NK细胞。具体到T细胞，富集后，将iHSC、MPP细胞转移到不含滋养层细胞的悬浮培养或含有OP9基质细胞或基质胶包覆表面的贴壁培养。对培养物补充含有可溶性DLL1和DLL4的iTC-A。大约10天后，将培养环境置换为iTC-B以完成T细胞成熟。大约30-40天(原始诱导分化后)，评估T细胞的组成，包括CD3，CD7，TCRαβ，CD4和CD8的表面表达。在一些实例中，得到的CD34阳性细胞产生通过来自CD7群体的CD4和CD8的表达所定义的不同T细胞群体的体外分化能力。具体到NK细胞，HSC细胞用包括IL15、iNK-A培养基的分化培养基处理大约10天，并切换到iNK-B培养基另外的10-20天。培养以悬浮形式进行。
本文描述的多阶段培养平台显示了使用顺序分化方法从多种干细胞中得到造血干细胞的过程。得到的CD34阳性造血干细胞可以保留在悬浮培养物中用于扩增(scaling)，并产生多种造血细胞谱系，包括造血干细胞、MPP、造血祖细胞、T细胞和NK细胞。此外，得到的CD34阳性永久造血干细胞显示通过上调免疫调节表面蛋白PDL1来响应药理学调节。此外，当植入时，得到的CD34阳性细胞能够在体内重建包括骨髓和淋巴群体。来自各种群体(包括多能干细胞)的永久造血干细胞是患者特异性治疗和再生医学应用的理想候选者。
hiPSC培养平台
1.iHSC平台
本申请提供了用于从多能干细胞包括iPSC获得中胚层细胞的培养基iHSC-A。在一些实例中，iPSC是初始iPSC。
在一些实例中，培养基包含GSK3β抑制剂和小分子SB4。在一些实例中，培养基包含 GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、bFGF、LIF、Activin A。在一些实例中，培养基中的GSK3β抑制剂能够激活Wnt信号通路。在一些实例中，GSK3β抑制剂是CHIR99021、BIO或肯帕罗酮。在一些实例中，GSK3β抑制剂是CHIR99021。在一些实例中，培养基iHSC-A包含胞外基质蛋白。在一些实例中，胞外基质蛋白包括matrigel、VTN-N、Laminin-511、Laminin-521或其组合。在本申请提供的具体实施例中，本文的培养基iHSC-A包含浓度范围如表1所示的基本培养基、附加培养基、小分子和/或细胞因子。在一些实例中，TRA-1-85在中胚层定向分化过程中稳定高表达。在一些实例中，SSEA4的膜表达在中胚层定向分化过程中逐步降低。SSEA4逐步降低标明诱导多能干细胞正逐渐分化而失去多能性。在一些实例中，CD56的表达在中胚层定向分化过程中呈现具备初始基准表达水平，随后降低又恢复甚至呈现高表达水平的状态。这说明在定向造血相关中胚层分化过程中，CD56在干细胞状态下普遍低表达，随着分化的进行CD56呈现先降后升，至中胚层时呈现高表达水平的动态变化过程。利用培养基iHSC-A诱导分化得到的中胚层可以有效地通过多种方法分化为造血内皮。
本申请提供了用于从中胚层细胞获得造血内皮的培养基iHSC-B。在一些实例中，培养基包含高浓度VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂。在一些实例中，培养基包含高浓度VEGF和/或VEGF通路激活剂、cAMP通路激活剂和iHSC-B基本培养基，且该培养基不含或基本上不含GSK3β抑制剂、Activin A、bFGF。在一些实例中，iHSC-B基本培养基是StemPro34培养基。在一些实例中，培养基中包含30-200ng/ml VEGF，优选地，50-200ng/ml VEGF，更优选地，100-200ng/ml VEGF。在一些实例中，VEGF通路激活剂是Deoxyshikonin。在一些实例中，VEGF通路激活剂是Isoarnebin I。在一些实例中，VEGF通路激活剂是Isoarnebin 4。在一些实例中，cAMP通路激活剂是Forskolin。在一些实例中，cAMP通路激活剂是PACAP 1-38。在一些实例中，iHSC-B包含Forskolin和VEGF。在一些实例中，iHSC-B包含Forskolin和Deoxyshikonin。在一些实例中，iHSC-B包含Forsklin和Isoarnebin 4。在一些实例中，iHSC-B包含Forskolin和Isoarnebin I。在一些实例中，培养基iHSC-B包含胞外基质蛋白。在一些实例中，胞外基质蛋白包括matrigel、VTN-N、Laminin-511、Laminin-521或其组合。在本申请提供的具体实施例中，本文的培养基iHSC-B包含浓度范围如表2所示的基本培养基、附加培养基、小分子和/或细胞因子。多种诱导分化方法来源的造血相关中胚层细胞均能通过iHSC-B培 养获得造血内皮。利用培养基iHSC-B诱导分化得到的造血内皮可以有效地通过多种方法分化为造血干细胞。
本申请提供了用于从造血内皮获得造血干细胞的培养基iHSC-C。在一些实例中，培养基iHSC-C包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选地造血干细胞促增殖分子。在一些实例中，培养基iHSC-C包含Notch信号通路激活剂、TGFβ受体/ALK抑制剂和iHSC-C基本培养基，且该培养基可选的包含造血干细胞促增殖分子，但不含或基本上不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂。在一些实例中，iHSC-C基本培养基是StemPro34培养基。在一些实例中，Notch信号通路激活剂是Resveratrol。在一些实例中，Notch信号通路激活剂是VPA。在一些实例中，TGFβ受体/ALK抑制剂是SB431542。在一些实例中，TGFβ受体/ALK抑制剂是E-616452。在一些实例中，TGFβ受体/ALK抑制剂是A83-01。在一些实例中，造血干细胞促增殖分子是UM729。在一些实例中，造血干细胞促增殖分子是UM171。在一些实例中，造血干细胞促增殖分子是SR1。在一些实例中，iHSC-C包含Resveratrol、SB431542和UM729。在一些实例中，培养基iHSC-C包含胞外基质蛋白。在一些实例中，胞外基质蛋白包括matrigel、VTN-N、Laminin-511、Laminin-521或其组合。在本申请提供的具体实施例中，本文的培养基iHSC-C包含浓度范围如表3所示的基本培养基、附加培养基、小分子和/或细胞因子。多种诱导分化方法来源的造血内皮均能通过iHSC-C培养获得造血干细胞。利用培养基iHSC-C诱导分化得到的造血干细胞可以有效地通过多种方法分化为T细胞、NK细胞。
本申请提供了用于从造血相关的中胚层细胞获得造血干细胞的培养基iHSC-D。在一些实例中，培养基iHSC-D包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂、TGFβ受体/ALK抑制剂和cAMP通路激活剂。在一些实例中，培养基iHSC-D包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂、TGFβ受体/ALK抑制剂和cAMP通路激活剂和iHSC-D基本培养基，且该培养基可选的包含造血干细胞促增殖分子，但不含或基本上不含bFGF、GSK3β抑制剂和/或BMP激活剂。在一些实例中，培养基iHSC-D包含30-200ng/ml VEGF，优选地，50-200ng/ml VEGF，更优选地，100-200ng/ml VEGF。在一些实例中，VEGF通路激活剂是Deoxyshikonin。在一些实例中，VEGF通路激活剂是Isoarnebin I。在一些实例中，VEGF通路激活剂是Isoarnebin 4。在一些实例中，cAMP通路激活剂是Forskolin。在一些实例中，cAMP通路激活剂是PACAP 1-38。在一些实例中，Notch信号通路激活剂是Resveratrol。在一些实例中，Notch信号通路激活剂是VPA。在一些实例中，TGFβ受体/ALK抑制剂是SB431542。在一些实例中，TGFβ受体/ALK抑制剂是E-616452。在一些实例中，TGFβ受体/ALK抑制剂是A83-01。在一些实例中，造血干细胞促增殖分子是UM729。在一些实例中，造血干细胞促增殖分子是UM171。在一些实例中，造血干细胞促增殖分子是SR1。在一些实例中，iHSC-D(VEGF)包含Forskolin、Resveratrol、SB431542和VEGF。在一些实例中，iHSC-D(Deoxyshikonin)包含Forskolin、Resveratrol、SB431542和Deoxyshikonin。在一些实例中，iHSC-D(Isoarnebin I)包含Forskolin、Resveratrol、SB431542和Isoarnebin I。在一些实例中，iHSC-D(Isoarnebin4)包含Forskolin、Resveratrol、SB431542和Isoarnebin 4。在一些实例中，iHSC-D基本培养基是StemPro34培养基。在一些实例中iHSC-D基本培养基是MCH5100培养基。在一些实例中，iHSC-D基本培养基是Stemspan II培养基。在一些实例中，培养基iHSC-D包含胞外基质蛋白。在一些实例中，胞外基质蛋白包括matrigel、VTN-N、Laminin-511、Laminin-521或其组合。在一些实例中，本文的培养基iHSC-D包含浓度范围如表4所示的基本培养基、附加培养基、小分子和/或细胞因子。多种诱导分化方法来源的造血相关中胚层细胞均能通过iHSC-D培养获得造血干细胞。利用培养基iHSC-D诱导分化得到的造血干细胞可以有效地通过多种方法分化为T细胞、NK细胞。
本申请的另一方面提供了用于获得T细胞的培养平台，其包含一种或多种(i)培养基， 其包含一种或多种选自SCF、Flt3L、IL7和IGF的生长因子和细胞因子和iTC基本培养基，后者包含一种或多种选自IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种Notch途径激活剂，其中该培养基不含BMP激活剂并适于从T祖细胞产生T细胞；(ii)培养基，其包含BMP激活剂，一种或多种选自SCF、Flt3L和IL7的生长因子和细胞因子和iTC基本培养基，其中培养基适于从iHSC产生T祖细胞。
本申请的另一方面提供了用于获得T细胞的培养平台，其包含一种或多种(i)培养基，其包含一种或多种选自SCF、Flt3L、IL7和IGF的生长因子和细胞因子和iTC基本培养基，后者包含一种或多种选自IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种Notch途径激活剂，其中该培养基不含BMP激活剂并适于从T祖细胞产生T细胞；(ii)培养基，其包含BMP激活剂，一种或多种选自SCF、Flt3L和IL7的生长因子和细胞因子和iTC基本培养基，其中培养基适于从iHSC产生T祖细胞。
本申请的一个方面提供了用于获得T祖细胞的培养平台，其包含一种或多种(i)培养基，其包含BMP激活剂，一种或多种选自SCF、Flt3L和IL7的生长因子和细胞因子和iTC基本培养基，其中培养基适于从iHSC产生T祖细胞。
在一实例中，用于获得NK细胞的培养平台包含(i)培养基，其包含一种或多种选自SCF、Flt3L、IGF和IL7的生长因子和细胞因子和iNK基本培养基，后者包含一种或多种选自IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种Notch途径激活剂，其中该培养基不含BMP激活剂并适于从NK祖细胞产生NK细胞。在一实例中，包含培养基(i)的培养平台进一步包含(ii)培养基，其包含BMP激活剂，一种或多种选自SCF、Flt3L和IL7的生长因子和细胞因子和iNK基本培养基，其中培养基(ii)适于从iHSC产生NK祖细胞。
本申请的一个方面提供了用于获得iHSC的培养平台，其包含一种或多种(i)培养基，其包含一种或多种选自SCF、GM-CSF、IL3、IL6、FLT-3L和TPO的细胞因子和iHSC-C基本培养基，后者包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，且该培养基可选的包含造血干细胞促增殖分子，但不含或基本上不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂，所述培养基适于从造血内皮产生iHSC；(ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、cAMP通路激活剂和iHSC-B基本培养基，且该培养基不含或基本上不含GSK3β抑制剂、Activin A和/或bFGF，所述培养基适于从中胚层细胞产生造血内皮；以及(iii)培养基，其包含GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、Activin A、bFGF和/或LIF，所述培养基适于从iPSC产生中胚层细胞。在一些实例中，iPSC是初始iPSC。在一些实例中，初始iPSC铺板密度约2.5x104cells/cm2。在一些实例中，初始iPSC铺板密度约10x104cells/cm2。在一些实例中，造血干细胞最早可于造血分化后第8天出现。在一些实例中，造血干细胞最早可于造血分化后第8天出现，且可持续收集造血干细胞至第20天。在一些实例中，24孔板每孔可单次收集诱导的造血干细胞量达到106级，且可以高增殖率的多代传代扩增。在一些实例中，iHSC平台制备的iHSC继续分化为T、NK细胞。在一些实例中，iHSC-B为 VEGF+Forskolin的组合时，iHSC平台可以获得阳性率高达80％的CD34+iHSC细胞。在一些实例中，iHSC-B分别VEGF+Forskolin、Deoxyshikonin+Forskolin、Isoarnebin I+Forskolin、Isoarnebin 4+Forskolin组合的培养条件下，iHSC培养平台1分化获得的HE占比约80％，动静脉内皮(主要是动脉内皮)占比约20％。这大大有利于高效的内皮造血转换，为后续的高效的分化获得诱导性造血干细胞奠定了坚实的平台基础。在一些实例中，不同的ECM培养条件下，iHSC平台能够稳定、高效地分化获得CD43+CD45+iHSC。由于CD38在iHSC和早期淋系都是阴性表达，在早期髓系是阳性表达。在一些实例中，在iHSC-B分别为VEGF+Forskolin、Deoxyshikonin+Forskolin、Isoarnebin I+Forskolin、Isoarnebin 4+Forskolin组合的培养条件下，iHSC平台能够稳定、高效地分化获得CD43+CD45+iHSC，其中，CD38+髓系类型细胞的占比仅不到1％，这说明本iHSC平台可用于髓系分化和/或淋系分化，或优先用于淋系分化。
本申请提供了用于获得iHSC的培养平台，其包含一种或多种(i)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂、TGFβ受体/ALK抑制剂和cAMP通路激活剂和iHSC-D基本培养基，且该培养基可选的包含造血干细胞促增殖分子，但不含或基本上不含bFGF、GSK3β抑制剂和/或BMP激活剂，所述培养基适于从中胚层细胞产生iHSC；以及(ii)培养基，其包含GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、Activin A、bFGF和/或LIF，所述培养基适于从iPSC产生中胚层细胞。在一些实例中，iPSC是初始iPSC。在一些实例中，初始iPSC铺板密度约2.5x104cells/cm2。在一些实例中，初始iPSC铺板密度约10x104cells/cm2。在一些实例中，造血干细胞最早可于造血分化后第8天出现。在一些实例中，造血干细胞最早可于造血分化后第9天出现，且可持续收集造血干细胞至第20天。在一些实例中，24孔板每孔可单次收集诱导的造血干细胞量达到106级，且可以高增殖率的多代传代扩增。在本申请提供的具体实施例中，iHSC-D基本培养基为MCH5100的iHSC平台制备的iHSC更倾向于或更有利于分化为巨噬细胞或单核细胞。在本申请提供的具体实施例中，iHSC-D基本培养基为StemSpan SFEM II或StemPro34的iHSC平台制备的iHSC更倾向于或更有利于分化为T、NK细胞。在一些实例中，iHSC-D基本培养基为MCH5100的iHSC平台制备的iHSC继续分化为巨噬细胞或单核细胞。在一些实例中，iHSC-D基本培养基为StemSpan SFEM II或StemPro34的iHSC平台制备的iHSC继续分化为T、NK细胞。在一些实例中，不同的ECM培养条件下，iHSC平台能够稳定、高效地分化获得CD43+CD45+iHSC。同时，由于CD38在iHSC和早期淋系都是阴性表达，在早期髓系是阳性表达。在一些实例中，iHSC-D基本培养基为MyeloCult H5100的培养条件下，iHSC平台能够稳定、高效地分化获得CD43+CD45+iHSC，其中，CD38+髓系类型细胞占比约20％，这说明该培养平台获得的iHSC有相对明显的早期髓系分化倾向，故该培养平台可用于髓系分化和/或淋系分化，或优选用于髓系分化。在一些实例中，iHSC-D基本培养基为StemPro34、StemSpan SFEM II的培养条件下，iHSC平台能够稳定、高效地分化获得CD43+CD45+iHSC，其中，CD38+ 髓系类型细胞的占比约0％～8％，这说明该培养平台可用于髓系分化和/或淋系分化，或优先用于淋系分化。
在一些实例中，用于获得iHSC的培养平台包含(i)培养基，其包含一种或多种选自SCF、GM-CSF、IL3、IL6、FLT-3L和TPO的细胞因子和iHSC-C基本培养基，后者还包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，且该培养基可选的包含造血干细胞促增殖分子，但不含或基本上不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂，所述培养基适于从造血内皮产生iHSC。在一些实例中，包含培养基(i)的培养平台进一步包含(ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、cAMP通路激活剂和iHSC-B基本培养基，且该培养基不含或基本上不含GSK3β抑制剂、Activin A和/或bFGF，所述培养基适于从中胚层细胞产生造血内皮。在一些实例中，包含培养基(i)的培养平台进一步包含(iii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂、TGFβ受体/ALK抑制剂和cAMP通路激活剂和iHSC-D基本培养基，且该培养基可选的包含造血干细胞促增殖分子，但不含或基本上不含bFGF、GSK3β抑制剂和/或BMP激活剂，所述培养基适于从中胚层细胞产生iHSC。在另一实施方式中，包含培养基(i)和(ii)的培养平台，进一步包含(iv)培养基，其包含GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、Activin A、bFGF和/或LIF，所述培养基适于从iPSC产生中胚层细胞。在另一实施方式中，包含培养基(i)和(iii)的培养平台，进一步包含(iv)培养基，其包含GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、Activin A、bFGF和/或LIF，所述培养基适于从iPSC产生中胚层细胞。在一些实例中，iPSC是初始iPSC。
本申请提供了用于获得造血内皮的培养平台，其包含一种或多种(i)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、cAMP通路激活剂和iHSC-B基本培养基，且该培养基不含或基本上不含GSK3β抑制剂、Activin A和/或bFGF，所述培养基适于从中胚层细胞产生造血内皮,以及(ii)培养基，其包含GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、Activin A、bFGF和/或LIF，所述培养基适于从iPSC产生中胚层细胞。
在一些实例中，用于获得造血内皮的培养平台包含(i)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、cAMP通路激活剂和iHSC-B基本培养基，且该培养基不含或基本上不含GSK3β抑制剂、Activin A和/或bFGF，所述培养基适于从中胚层细胞产生造血内皮。在另一实施方式中，包含培养基(i)的培养平台，进一步包含(ii)培养基，其包含GSK3β抑制剂和小分子SB4和iHSC-A基本培养基，且该培养基不含或基本上不含BMP4、Activin A、bFGF和/或LIF，所述培养基适于从iPSC产生中胚层细胞。
培养基iTC-A包含BMP激活剂，一种或多种选自SCF、Flt3L、IL7、IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种选自Jag1、Jag2、DLL-1、DLL-3和DLL-4的Notch途径激活剂；在一些实例中，该组合物不含VEGF和/或IL15。培养基iTC-B包含一种或多 种选自SCF、Flt3L、IL7、IGF、IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种选自Jag1、Jag2、DLL-1、DLL-3和DLL-4的Notch途径激活剂；在一些实例中，该组合物不含BMP激活剂。培养基iNK-A包含BMP激活剂，一种或多种选自SCF、Flt3L、VEGF、IL2、IL3、IL6和IL15的生长因子和细胞因子。培养基iNK-B包含一种或多种选自SCF、Flt3L、IGF、IL7、IL2、IL3、IL6和IL15的生长因子。
基于本申请的教导，本领域技术人员应该理解，本申请具体公开的剂量是本申请人经过研究得到是安全有效的剂量，本领域技术人员，例如临床医师可以根据各种实际情况，例如患者的肿瘤负荷、患者本身的身体状况等因素决定每个周期的给药剂量。
本申请的优点：
本申请提供了能够使多能干细胞单层直接分化而不从多能干细胞产生拟胚体的方法和组合物，从而能够进行中胚层细胞、HE和iHSC、T细胞、NK细胞的分化和扩增，其中其它造血细胞谱系能够以非常高的效率水平以规模化的、可靠的模式获得。
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。本说明书中提到的所有出版物、专利和专利申请均通过引用并入本文，其程度如同特别地且单独地指出每一个单独的出版物、专利或专利申请均通过引用而并入本文。
实施例
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。
实施例1.人诱导多能干细胞(hiPSCs)产生和维持
示例性的，在阶段性存在含有ROCK、HADC、GSK3β途径和TGFβ受体抑制剂，以及cAMP激活剂的重编程培养基中，使用包括Oct4/Sox2/Klf4或Oct4/Sox2/Klf4/mp53DD/EBNA1或Oct4/Sox2/Klf4/Lin28/L-Myc/mp53DD/EBNA1在内的各种因子组合诱导包括人非动员外周血CD34+干细胞、外周血内皮前体细胞在内的体细胞向多能状态重新编程。诱导后14-16天后，逐步挑取状态良好的重编程获得的hiPSCs单克隆作为初始细胞，移入matrigel、VTN或LN等作为细胞外基质、mTeSR1Plus或E8medium等作为维持培养基的诱导多能干细胞培养体系中，进行驯化扩增培养。
经过至少10代的稳定驯化后，进行相应的系列干细胞多能性、安全性等检测实验(包括不限于检测细胞内的转基因残留或潜在基因组插入风险，检测SNP、核型、支原体/细菌/病毒/内毒素等，进行多能性基因的复合表达检测，检测体内外三胚层的分化发育潜能等等)。所获得的hiPSC为初始/起始细胞。
实施例2.使用iHSC培养平台1的造血分化
为了启动向造血细胞谱系的分化，将初始hiPSC以单层培养体系接种在维持培养基中 并允许扩张直到达到约25％的铺板面积。将维持培养基切换为如表1所示的培养基iHSC-A开始造血分化(记为第0天)。培养物随后在分化开始后第3-4天切换为如表2所示的培养基iHSC-B，在分化开始后的第5-20天切换为如表3所示的培养基iHSC-C。附着的培养物在培养基变化期间保持贴壁并且不受干扰。iHSC培养平台1是无血清无滋养层的单层培养的造血培养平台(图1)。
在分化过程早期(第0天至第3天)，通过谱系标志物CD56和BRACHYURY监测造血中胚层分化；在第4天(此时间点可以延长，最长至第8天)，通过CD31、CD144、CD34、CD184以及CD73的监测表达鉴别造血内皮的定向诱导，需要说明的是，伴随造血内皮的分化，动脉内皮的伴随分化是必要而有益的。在造血内皮诱导完成后，通过观察从变圆且有脱离贴壁形式的细胞的出现，至晶亮珠形圆球样造血干样细胞群体成簇或散在的逐步出现，基本可以从表型上确认已分化至造血干细胞阶段，同时在造血干细胞诱导分化接近完全时，收集细胞进行相应的标志基因类群检测(CD34、CD43、CD45、CD31、CD144、CD90、CD38、CD45RA)和造血分化潜能检测(CFU单克隆集落形成)。
实施例3.使用iHSC培养平台2的造血分化
为了启动向造血细胞谱系的分化，将初始hiPSC以单层培养体系接种在维持培养基中并允许扩张直到达到约25％的铺板面积。此时，通过将维持培养基切换为如表1所示的培养基iHSC-A开始造血分化。培养物随后在分化开始后第3天起切换为如表4所示的培养基iHSC-D。附着的培养物在培养基变化期间保持贴壁并且不受干扰。iHSC培养平台2是无血清无滋养层的单层培养的造血培养平台(图2)。
在分化过程早期(第0天至第3天)，通过谱系标志物CD56和BRACHYURY监测造血中胚层分化；在第4天至第5天(此时间段可在1天至4天内调整)，通过CD31、CD144、CD34、CD184以及CD73的监测表达鉴别造血内皮的定向诱导，需要说明的是，伴随造血内皮的分化，动脉内皮的伴随分化是必要而有益的。在造血内皮诱导完成后，通过观察从变圆且有脱离贴别形式的细胞的出现，至晶亮珠形圆球样造血干样细胞群体的或成簇或散在的逐步出现，基本可以从表型上确认已分化至造血干细胞阶段，同时在造血干细胞诱导分化接近完全时，收集细胞进行相应的标志基因类群检测(CD34、CD43、CD45、CD31、CD144、CD90、CD38、CD45RA)和造血分化潜能检测(CFU单克隆集落形成)。
实施例4.单层分化系统的造血中胚层定向分化
参照实施例1获得的hiPSCs为初始细胞。将hiPSCs铺入包被matrigel、VTN或LN的孔板内。通过将维持培养基切换为培养基iHSC-A开始造血分化(记为第0天)。造血分化第0天到第3天是中胚层形成阶段，实现向Brachyury+CD56+中胚层的定向分化(图3)。iHSC-A组成：一方面DMEM:F12(Gibco)添加Glutamax(Gibco)，另一方面Neurobasal media(Gibco)补充N2(Gibco)和B27(Gibco)，然后两者1：1混合配置；然后额外添加5-10μM CHIR99021和5-10μM SB 4(iHSC-A组)或5-10μM CHIR99021和12.5-25ng/ml BMP4(hiPSC-Pro组)。iHSC-A组继续诱导分化可高效获得造血干细胞，而在hiPSC-Pro组诱导分化的中胚层细胞 不能有效分化获得造血细胞。图4检测培养基iHSC-A诱导分化得到的中胚层细胞的TRA-1-85、SSEA4、CD56表达情况。
实施例5.造血内皮的诱导分化
获得造血相关中胚层后继续进行造血内皮的诱导分化。示例性的，接着实施例4，造血分化的第3天至第4天为造血内皮分化阶段(该阶段可持续2-4天)，利用VEGF结合bFGF的细胞因子组合，实现向CD31+(或CD144+)CD34+CD184+造血内皮的定向分化。
造血内皮诱导阶段为第3天到第4天，所用诱导培养液为EGM 2medium(Lonza)，额外添加50ng/ml VEGF，10ng/ml bFGF。经过2天的诱导，获得了具有造血分化潜能的内皮细胞群体。利用本申请的方法得到的中胚层可以有效地通过多种方法分化为造血内皮。
实施例6.造血内皮的诱导分化
获得造血相关中胚层后继续进行造血内皮的诱导分化。示例性的，接着实施例4，造血分化的第3-4天切换为培养基iHSC-B开始造血内皮分化阶段(该阶段可持续1-5天)，实现向CD31+CD34+CD184+、CD144+CD34+CD184+、CD31+CD144+CD34+CD184+造血内皮的定向分化。
示例性的，造血内皮诱导阶段在细胞外基质matrigel或VTN-N或laminin-511或laminin-521等的支持下，所用iHSC-B(VEGF+Forskolin)为添加100-200ng/ml VEGF和2-4μM Forskolin，和添加Glutamine的StemPro34培养基；或iHSC-B(Deoxyshikonin+Forskolin)为添加1-6μM Deoxyshikonin和2-4μM Forskolin，和添加Glutamine的StemPro34培养基；或iHSC-B(Isoarnebin I+Forskolin)为添加1-10μM Isoarnebin I和2-4μM Forskolin，和添加Glutamine的StemPro34培养基；或iHSC-B(Isoarnebin 4+Forskolin)为添加1-10μM Isoarnebin 4和2-4μM Forskolin，和添加Glutamine的StemPro34培养基。经过2天左右的诱导，获得了具有造血分化潜能的内皮细胞群体。图5A示例性的所用的iHSC-B(VEGF+Forskolin)、iHSC-B(Deoxyshikonin+Forskolin)。图5B、图5C示例性的所用的iHSC-B(VEGF+Forskolin)、iHSC-B(Deoxyshikonin+Forskolin)。
实施例7.造血干细胞的诱导分化
获得造血内皮后继续进行造血干细胞的诱导分化。示例性的，接着实施例5、或6，造血分化的第5天到第20天为造血干细胞分化阶段，利用多种细胞因子结合小分子SB431542的方式，实现向CD43+CD45+造血干细胞的定向分化。
造血干细胞诱导阶段为第5天至第20天左右，示例性的，所用培养液为STEMdiff APEL2medium(Stemcell)，额外添加50ng/ml SCF(Peprotech),50ng/ml FLT3-L(Peprotech),50ng/ml TPO(Peprotech),10ng/ml IL-3(Peprotech),10ng/ml VEGF(Peprotech),10ng/ml bFGF(Peprotech)和10μM SB-431542(Selleck)。经过6天左右的诱导，获得了大量的造血干细胞，可用于后期的免疫细胞定向分化(例如，CD4T细胞、CD8T细胞和NK细胞等)。可见利用本申请得到的造血内皮可以通过多种方法分化为造血干细胞。
实施例8.造血干细胞的诱导分化
获得造血内皮后继续进行造血干细胞的诱导分化。示例性的，接着实施例6，造血分化的第5天到第20天切换为培养基iHSC-C，开始造血干细胞分化阶段，实现向CD34+CD43+CD45+或CD43+CD45+造血干细胞的定向分化。
造血干细胞诱导阶段为第5天至第20天左右(造血干细胞的收集可持续至20天左右，可多次收集)。示例性的，在matrigel或VTN-N或laminin-511或laminin-521等的支持下，iHSC-C为StemPro34(Gibco)，额外添加2mM GluMAX(Gibco)，100ng/ml SCF，25ng/ml GM-CSF，50ng/ml IL-3，0-100ng/ml IL-6，50-100ng/ml FLT-3L，50-100ng/ml TPO，25-50ng/ml ascorbic acid，5-10μM Resveratrol，5-10μM SB431542，以及可以额外加入1-2μM UM729，经过5-10天左右的诱导(可持续诱导多次收集,至孔内不再残存剩余非造血干细胞为止)，获得了大量的造血干细胞(图6)，可用于后期的免疫细胞定向分化(例如，CD4T细胞、CD8T细胞和NK细胞等)。多种诱导分化方法来源的造血内皮均能通过iHSC-C培养获得造血干细胞。利用培养基iHSC-C诱导分化得到的造血干细胞可以有效地通过多种方法分化为T细胞、NK细胞。
实施例9.造血干细胞的诱导分化
获得造血相关中胚层后继续进行造血内皮的诱导分化。示例性的，接着实施例4，造血分化的第3天到第20天切换为培养基iHSC-D，开始造血干细胞分化阶段，实现向CD34+CD43+CD45+或CD43+CD45+造血干细胞的定向分化。
造血干细胞诱导阶段为第3天至第20天左右，(造血干细胞的收集可持续至20天左右,可多次收集)。示例性的，在matrigel或VTN-N或laminin-511或laminin-521等的支持下，iHSC-D基本培养基为StemPro34(Gibco)，额外添加2mM GluMAX(Gibco)，25-50ng/ml ascorbic acid；或iHSC-D基本培养基为MyeloCultTMH5100(stemcell)，额外添加25-50ng/ml ascorbic acid；或iHSC-D基本培养基为StemSpanTMSFEM II额外添加25-50ng/ml ascorbic acid。包括上述基本培养基的iHSC-D(VEGF)还添加100-200ng/ml VEGF、2-4μM forskolin、5-10μM Resveratrol和5-10μM SB431542。包括上述基本培养基的iHSC-D(Deoxyshikonin)还添加1-6μM Deoxyshikonin、2-4μM forskolin，5-10μM Resveratrol和5-10μM SB431542。包括上述基本培养基的iHSC-D(Isoarnebin I)还分别添加1-10μM Isoarnebin I、2-4μM forskolin、5-10μM Resveratrol和5-10μM SB431542。包括上述基本培养基的iHSC-D(Isoarnebin 4)还分别添加1-10μM Isoarnebin 4、2-4μM forskolin、5-10μM Resveratrol和5-10μM SB431542。经过至少7-12天左右的诱导(可持续诱导多次收集,至孔内不再残存剩余非造血干细胞为止)，获得了大量的造血干细胞(图7)，可用于后期的免疫细胞定向分化(例如，CD4T细胞、CD8T细胞和NK细胞等)。多种诱导分化方法来源的造血相关中胚层细胞均能通过iHSC-D培养获得造血干细胞。利用培养基iHSC-D诱导分化得到的造血干细胞可以有效地通过多种方法分化为T细胞、NK细胞。
实施例10 hiPSC不同铺板密度下的造血分化检测
在Matrigel涂覆的培养皿上，hiPSC的量从低的铺板密度约2.5x104cells/cm2增加至高的 铺板密度约10x104cells/cm2/孔，进行造血分化检测(图8)。
实施例11不同ECM涂覆情况下的造血分化检测
在matrigel、VTN-N或laminin-511分别涂覆的培养皿中，hiPSC通过iHSC平台1进行造血分化检测(图9)；hiPSC通过iHSC平台2进行造血分化检测(图10)。
实施例12.iHSC的造血潜能的检测
利用CFU单克隆集落形成实验检测所获得的iHSC的造血潜能。利用MethoCult H4034Optimum(STEMCELL Technologies)混合的半固体培养基混合所获得的iHSC。然后将混合物转移到孔板中，37℃、5％CO2、100％湿度下孵育14天，然后观察菌落。每种菌落根据形态进行分类(如图11)，观察记录到:CFU-GM、CFU-G、CFU-MK、BFU-E、CFU-GEMM等多种谱系克隆类型的出现，表征该iHSC具有相对全能的造血分化能力。
同时，利用流式细胞检测分析了获得的iHSC自发分化的髓淋系倾向，如图12，造血定向分化获得的iHSC，具备分别代表了髓系分化倾向和淋系分化倾向的CD14+和CD45RA+两种阳性类群。这证明了分化获得的iHSC的多潜能造血分化能力。
实施例13.使用iHSC培养平台进行分化后特定造血谱系的继续分化
分选或富集的iHSC细胞沿着造血谱系进一步分化为各种特定细胞类型，包括T细胞和NK细胞。
示例性的iHSC进一步分化到T细胞。所富集的iHSC细胞加入在用vitronectin和hDLL-4/Fc包被好的孔板中，孵育培养基组成：StemSpan SFEM II,100×ITS-G(1×)，55μM2-Mercaptoethanol,50μg/ml Vc,Glutamax 2Mm,50ng/ml rHSCF、50ng/ml rhTPO、50ng/ml rhIL-7、50ng/ml FLT3L、30nM rhSDF-1α和15μM SB203580，培养约28天后，将细胞移入新包被的孔板中，加入培养基组成：StemSpan SFEM II,1×ITS-G，50μg/ml抗坏血酸-2-phosphate 1×PSG,10ng/ml rhIL-7,10ng/ml rhIL-2,和10nM地塞米松，持续培养14天。细胞激活利用CD3/CD28beads。收集细胞利用CD3/CD8/CD4/CD45等T细胞标志物进行检测(如图13)。
示例性的iHSC进一步分化到NK细胞。所富集的iHSC细胞与OP9-DL1细胞进行共培养，培养基为StemSpan SFEM II，300IU/mL IL-2，50ng/mL IL-12，50ng/ml IL-18，50ng/ml IL-21，50ng/ml IL27，50ng/ml IL-15，5％人血小板裂解物，5mmol/L烟酰胺(NAM)，5mg/ml Vitamin E和5μg/mL Heparin，培养约38天。收集细胞进行CD45/CD34/CD56/CD16流式染色，检测NK细胞比例。结果显示NK细胞比例最高可达95％以上。
以上所述仅是本申请的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本申请方法的前提下，还可以作出若干改进和补充，这些改进和补充也应视为本申请的保护范围。
本申请所述实施例包括将该实施例作为任何单一实施例或与任何其他实施例或其部分相结合。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (42)

  1. 诱导干细胞分化的方法，其特征在于，使多能干细胞与包括GSK3β抑制剂、以及下式I所示的化合物A的组合物接触以获得中胚层细胞，
    其中，R1选自：
    R2为
    其中，X4、X5、X6、或X7选自H、甲基、乙基、羟基、甲氧基、乙氧基、或卤素，X4、X5、X6、或X7可以相同，也可以不同；
    优选R2选自以下任一结构：
    所述中胚层细胞能够提供造血细胞谱系，所述造血细胞谱系包括造血内皮、造血干细胞和造血祖细胞、造血多能祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；且在中胚层诱导阶段不形成拟胚体。
  2. 如权利要求1所述的方法，其特征在于，进一步包括：使所述中胚层细胞接触包含VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂的组合物，以获得造血内皮。
  3. 如权利要求2所述的方法，其特征在于，进一步包括：使所述造血内皮接触包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的造血干细胞促增殖分子的组合物，以获得造血干细胞。
  4. 如权利要求1所述的方法，其特征在于，进一步包括：使所述中胚层细胞接触包含VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选 的cAMP通路激活剂的组合物，以获得造血干细胞。
  5. 如权利要求1-4任一所述的方法，进一步包括：使多能干细胞、中胚层细胞、造血内皮和/或造血干细胞处于低氧条件下。
  6. 如权利要求1-5任一所述的方法，其中所述干细胞分化过程不含BMP4、Activin A、bFGF和/或LIF。
  7. 如权利要求1-6任一所述的方法，其中，
    (i)所述干细胞分化处于无滋养层条件下；或
    (ii)所述干细胞分化处于有细胞外基质条件下；或
    (iii)多能干细胞包括诱导性多能干细胞(iPSC)，可选的iPSC是初始iPSC。
  8. 如权利要求1-7任一所述的方法，其特征在于，所述化合物A是SB4，其结构如下图所示：
  9. 如权利要求1-8任一所述的方法，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；或
    Notch信号通路激活剂是Resveratrol和/或VPA；或
    TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；或
    所述造血干细胞促增殖分子是UM729、UM171和/或SR1。
  10. 用于产生多能干细胞来源的中胚层细胞的方法，其特征在于，使多能干细胞接触包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4的组合物，以获得中胚层细胞；所述中胚层细胞能够提供造血细胞谱系，所述造血细胞谱系包括造血内皮、造血干细胞和造血多能祖细胞、造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；且在中胚层诱导阶段不形成拟胚体。
  11. 用于产生多能干细胞来源的造血内皮的方法，其特征在于，使多能干细胞来源的中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂的组合物，以产生造血内皮，所述造血内皮能够提供造血细胞谱系：包括造血干细胞、造血多能祖细胞、造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；且在造血内皮诱导阶段不形成拟胚体。
  12. 如权利要求11所述的方法，其特征在于，进一步包括使多能干细胞接触包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4的组合物，以获得中胚层细胞，优选由多能干细胞分化为中胚层细胞的过程如权利要求1-9中任一项所述。
  13. 用于产生多能干细胞来源的造血干细胞的方法，其特征在于，包括：
    方法I：使多能干细胞来源的造血内皮接触包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的造血干细胞促增殖分子的组合物，以产生造血干细胞；或
    方法II：使多能干细胞来源的中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的cAMP通路激活剂的组合物，以产生造血干细胞，
    在产生造血干细胞的过程中不形成拟胚体；
    优选由权利要求1-9中任一项所述方法获得多能干细胞来源的中胚层细胞；优选由权利要求10-12任一所述方法获得多能干细胞来源的造血内皮；。
  14. 如权利要求10-13任一项所述的方法，
    (i)所述用于产生多能干细胞来源的中胚层细胞的方法或用于产生多能干细胞来源的造血内皮的方法或用于产生多能干细胞来源的造血干细胞的方法不含BMP4、Activin A、bFGF和/或LIF；或
    (ii)使多能干细胞、中胚层细胞、造血内皮和/或造血干细胞处于低氧条件下；或
    (iii)所述用于产生多能干细胞来源的中胚层细胞的方法或用于产生多能干细胞来源的造血内皮的方法或用于产生多能干细胞来源的造血干细胞的方法涉及的细胞分化处于无滋养层条件下；或
    (iv)所述用于产生多能干细胞来源的中胚层细胞的方法或用于产生多能干细胞来源的造血内皮的方法或用于产生多能干细胞来源的造血干细胞的方法涉及的细胞分化处于有细胞外基质条件下；或
    (v)多能干细胞包括诱导性多能干细胞(iPSC)，可选的iPSC是初始iPSC。
  15. 如权利要求10-14任一所述的方法，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；或
    Notch信号通路激活剂是Resveratrol和/或VPA；或
    TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；或
    所述造血干细胞促增殖分子是UM729、UM171和/或SR1。
  16. 用于产生多能干细胞来源的造血多能祖细胞(MPP)的方法，包括：
    方法I包括：
    (i)使造血内皮接触包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的造血干细胞促增殖分子，以及一种或多种选自SCF、GM-CSF、IL-3、IL-6、FLT-3L和TPO的生长因子和细胞因子的组合物，以获得MPP，其中，组合物可选的不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂；
    (ii)其中所述造血内皮获得自使中胚层细胞接触包含VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂的组合物，以允许细胞分化和扩增，其中，组合物可选的不含 GSK3β抑制剂、Activin A和/或bFGF；
    (iii)其中所述中胚层细胞获得自使多能干细胞接触包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4的组合物，以允许细胞分化和扩增，其中组合物不含BMP4、Activin A、bFGF和/或LIF；或
    方法II包括：
    (i)使中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的cAMP通路激活剂的组合物，以获得MPP，其中，组合物可选的不含bFGF、GSK3β抑制剂和/或BMP激活剂；
    (ii)其中所述中胚层细胞获得自使多能干细胞接触包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4的组合物，以允许细胞分化和扩增，其中组合物不含BMP4、Activin A、bFGF和/或LIF；
    所述MPP能提供如下细胞：造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞。
  17. 如权利要求16所述的方法，其中，
    (a)使接种的多能干细胞、中胚层细胞、造血内皮和/或MPP处于约低氧条件下；或
    (b)其中多能干细胞来源的MPP诱导阶段(i)不形成拟胚体；(ii)在单层培养下；(iii)在无滋养层条件下；或(iv)在有细胞外基质条件下；或
    (c)其中多能干细胞是iPSC或初始iPSC。
  18. 如权利要求16或17所述的方法，其中
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；或
    Notch信号通路激活剂是Resveratrol和/或VPA；或
    TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；或
    造血干细胞促增殖分子是UM729、UM171和/或SR1。
  19. 用于引导人的多能干细胞分化成造血细胞谱系的方法，包括：
    方法I包括：
    (i)使多能干细胞接触包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4的组合物，以获得中胚层细胞，其中组合物不含BMP4、Activin A、bFGF和/或LIF；
    (ii)使中胚层细胞接触包含VEGF和/或VEGF通路激活剂的组合物，以及可选的cAMP通路激活剂的组合物，以获得造血内皮，其中组合物可选的不含GSK3β抑制剂、Activin A和/或bFGF；以及
    (iii)使造血内皮接触包含一种或多种选自SCF、GM-CSF、IL-3、IL-6、FLT-3L和TPO的生长因子和细胞因子；以及Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选 的造血干细胞促增殖分子的组合物，以获得造血干细胞，其中组合物可选的不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂；或；
    方法II包括：
    (i)使多能干细胞接触包含GSK3抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4的组合物，以获得中胚层细胞，其中组合物不含BMP4、Activin A、bFGF和/或LIF；
    (ii)使中胚层细胞接触包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的cAMP通路激活剂的组合物,以获得造血干细胞，其中组合物可选的不含bFGF、GSK3β抑制剂和/或BMP激活剂；
    所述造血干细胞能提供造血细胞谱系，所述造血细胞谱系包括：MPP、造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞。
  20. 如权利要求19所述的方法，其中，
    (a)使接种的多能干细胞、中胚层细胞、造血内皮和/或造血干细胞处于低氧条件下；或
    (b)其中造血细胞谱系诱导阶段(i)不形成拟胚体；(ii)在单层培养下；(iii)在无滋养层条件下；或(iv)在有细胞外基质条件下；或
    (c)其中多能干细胞是iPSC或初始iPSC。
  21. 如权利要求19或20所述的方法，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；或
    Notch信号通路激活剂是Resveratrol和/或VPA；或
    TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；或
    造血干细胞促增殖分子是UM729、UM171和/或SR1。
  22. 一种促进造血自我更新、重建或移植的方法，其使用包含一种或多种使用权利要求1-21任一项所述的方法产生的细胞谱系、细胞群或克隆细胞的组合物，其中，
    所述细胞谱系、细胞群克隆细胞选自如下中的任意：
    (i)多能干细胞来源的Brachyury+CD56+中胚层细胞，所述中胚层细胞能够分化为造血内皮、造血干细胞、MPP、造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；
    (ii)多能干细胞来源的造血内皮(iHE)，所述iHE是CD31+CD34+CD184+或CD144+CD34+CD184+或CD34+CD144+CD34+CD184+；
    (iii)多能干细胞来源的造血干细胞(iHSC),所述iHSC是CD34+CD43+CD45+或CD43+CD45+；
    (iv)多能干细胞来源的造血多能祖细胞(iMPP)，其中iMPP细胞是CD34+CD43+CD45+ 或CD43+CD45+；
    (v)多能干细胞来源的T祖细胞，其中T祖细胞是CD2+CD44+或CD25+CD44+；
    (vi)多能干细胞来源的T细胞，其中T细胞是CD4+或CD8+；
    (vii)多能干细胞来源的NK祖细胞，其中NK祖细胞是CD122+；和
    (viii)多能干细胞来源的NK细胞，其中NK细胞是CD56+CD16+。
  23. 一种用于获得多能干细胞来源的造血细胞谱系而不产生拟胚体的培养平台或用于获得和扩增多能干细胞来源的造血细胞谱系的组合物，包含：
    平台I或组合物I包括：
    (i)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的造血干细胞促增殖分子，以及一种或多种选自SCF、GM-CSF、IL-3、IL-6、FLT-3L和TPO的生长因子和细胞因子，其中该培养基可选的不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂，并适于从造血内皮分化和扩增造血干细胞；
    (ii)培养基，其包含VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂，其中该培养基适于从中胚层细胞分化和扩增所述造血内皮，其中组合物可选的不含GSK3β抑制剂、Activin A和/或bFGF；和
    (iii)培养基，其包含GSK-3抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4，其中组合物不含BMP4、Activin A、bFGF和/或LIF，其中该培养基适于从多能干细胞分化和扩增所述中胚层细胞；或，
    平台II或组合物II包括：
    (i)培养基，其包含VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的cAMP通路激活剂，其中该培养基可选的不含bFGF、GSK3β抑制剂和/或BMP激活剂，且其中该培养基适于从中胚层细胞分化和扩增造血干细胞；
    (ii)培养基，其包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4，其中组合物不含BMP4、Activin A、bFGF和/或LIF，其中该培养基适于从多能干细胞分化和扩增所述中胚层细胞，
    所述造血干细胞能提供造血细胞谱系，所述造血细胞谱系包括：MPP、造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞。
  24. 如权利要求23所述的培养平台或组合物，其中，
    (a)所述平台或组合物进一步包括添加ROCK抑制剂的维持培养基，适于接种和扩增多能干细胞；或
    (b)其中多能干细胞是iPSC或初始iPSC。
  25. 如权利要求23或24所述的培养平台或组合物，其中
    GSK3抑制剂是CHIR99021、BIO和/或肯帕罗酮；获
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；或
    Notch信号通路激活剂是Resveratrol和/或VPA；或
    TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；或
    所述造血干细胞促增殖分子是UM729、UM171和/或SR1。
  26. 一种用于产生多能干细胞来源的造血内皮的培养平台，包含：
    (i)培养基，其包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4，该培养基不含BMP4、Activin A、bFGF和/或LIF，该培养基适于从多能干细胞分化和扩增中胚层细胞；
    (ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂，该培养基可选的不含GSK3β抑制剂、Activin A和/或bFGF，该培养基适于从所述中胚层细胞分化和扩增造血内皮。
  27. 如权利要求26所述的培养平台，其中，
    (a)所述平台进一步包括添加ROCK抑制剂的维持培养基，适于接种和扩增多能干细胞；或
    (b)其中多能干细胞是iPSC或初始iPSC。
  28. 如权利要求26或27所述的培养平台，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38。
  29. 一种用于产生多能干细胞来源的中胚层细胞的培养平台，包含：
    (i)培养基，其包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4，其中该培养基适于从多能干细胞分化和扩增中胚层细胞；所述中胚层细胞能够提供造血细胞谱系：造血内皮、造血干细胞和造血祖细胞、MPP、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；且在中胚层诱导阶段不形成拟胚体。
  30. 如权利要求29所述的培养平台，其中，
    (a)所述平台进一步包括添加ROCK抑制剂的维持培养基，适于接种和扩增多能干细胞；或
    (b)其中多能干细胞是iPSC或初始iPSC。
  31. 如权利要求29或30所述的培养平台，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮。
  32. 一种用于产生多能干细胞来源的造血多能祖细胞(MPP)的培养平台，包括：
    平台I包括：
    (i)培养基，其包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的造血干细胞促增殖分子，以及一种或多种选自SCF、GM-CSF、IL-3、IL-6、FLT-3L和TPO的生长因子和细胞因子，其中该培养基可选的不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂，并适于从造血内皮分化和扩增MPP；
    (ii)培养基，其包含高浓度VEGF和/或VEGF通路激活剂，以及可选的cAMP通路激活剂，其中该培养基适于从中胚层细胞分化和扩增所述造血内皮，其中组合物可选的不含GSK3β抑制剂、Activin A和/或bFGF；和
    (iii)培养基，其包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4，其中组合物不含BMP4、Activin A、bFGF和/或LIF，其中该培养基适于从多能干细胞分化和扩增所述中胚层细胞；或，
    平台II包括：
    (i)培养基，其包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的cAMP通路激活剂，其中该培养基可选的不含bFGF、GSK3β抑制剂和/或BMP激活剂，且其中该培养基适于从中胚层细胞分化和扩增MPP；
    (ii)培养基，其包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4，其中组合物不含BMP4、Activin A、bFGF和/或LIF，其中该培养基适于从多能干细胞分化和扩增所述中胚层细胞。
  33. 如权利要求32所述的培养平台，其中，
    (a)所述平台进一步包括添加ROCK抑制剂的维持培养基，适于接种和扩增多能干细胞；或
    (b)其中多能干细胞是iPSC或初始iPSC。
  34. 如权利要求32或33所述的培养平台，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；Notch信号通路激活剂是Resveratrol和/或VPA；TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；造血干细胞促增殖分子是UM729、UM171和/或SR1。
  35. 一种组合物，包含：
    (a)一种或多种权利要求23-34任一所述的平台或组合物中涉及的培养基；以及
    (b)一种或多种从权利要求1-21任一所述的方法或权利要求23-34任一项所述的培养平台产生的细胞群，所述细胞群选自以下中的任意：
    (i)多能干细胞来源的Brachyury+CD56+中胚层细胞，所述中胚层细胞能够分化为造血内皮、造血干细胞、MPP、造血祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；
    (ii)多能干细胞来源的造血内皮(iHE)，所述iHE是CD31+CD34+CD184+或CD144+CD34+CD184+或CD34+CD144+CD34+CD184+；
    (iii)多能干细胞来源的造血干细胞(iHSC),所述iHSC是CD34+CD43+CD45+或CD43+CD45+；
    (iv)多能干细胞来源的造血多能祖细胞(iMPP)，其中iMPP细胞是CD34+CD43+CD45+ 或CD43+CD45+；
    (v)多能干细胞来源的T祖细胞，其中T祖细胞是CD2+CD44+或CD25+CD44+；
    (vi)多能干细胞来源的T细胞，其中T细胞是CD4+或CD8+；
    (vii)多能干细胞来源的NK祖细胞，其中NK祖细胞是CD122+；和
    (viii)多能干细胞来源的NK细胞，其中NK细胞是CD56+CD16+。
  36. 一种组合物，包含选自下述的多能干细胞来源的造血细胞谱系以及一种或多种培养基：
    (i)造血内皮(iHE)，以及一种或多种选自下述的培养基：iHSC-A、iHSC-B、iHSC-C、iHSC-D、iTC-A、iTC-B、iNK-A、iNK-B；
    (ii)造血干细胞，以及一种或多种选自下述的培养基：iHSC-A、iHSC-B、iHSC-C、iHSC-D、iTC-A、iTC-B、iNK-A、iNK-B；
    (iii)T祖细胞，以及一种或多种选自下述的培养基：iTC-A和iTC-B；
    (v)T细胞和iTC-B；
    (vi)NK祖细胞，以及一种或多种选自下述的培养基：iNK-A和iNK-B；以及
    (vii)NK细胞和iNK-B
    其中iHSC-A包含GSK3β抑制剂和权利要求1涉及的化合物A，优选权利要求8涉及的SB4；且不含BMP4、Activin A、bFGF和/或LIF；
    其中iHSC-B包含高浓度VEGF和/或VEGF通路激活剂的组合物，以及可选的cAMP通路激活剂；
    其中iHSC-C包含Notch信号通路激活剂和TGFβ受体/ALK抑制剂，以及可选的造血干细胞促增殖分子；
    其中iHSC-D包含高浓度VEGF和/或VEGF通路激活剂、Notch信号通路激活剂和TGFβ受体/ALK5抑制剂，以及可选的cAMP通路激活剂；
    其中iTC-A包含BMP激活剂，一种或多种选自SCF、Flt3L、IL7、IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种选自Jag1、Jag2、DLL-1、DLL-3和DLL-4的Notch途径激活剂；
    其中iTC-B包含一种或多种选自SCF、Flt3L、IL7、IGF、IL2、IL3和IL6的生长因子和细胞因子，以及一种或多种选自Jag1、Jag2、DLL-1、DLL-3和DLL-4的Notch途径激活剂；；
    其中iNK-A包含BMP激活剂，一种或多种选自SCF、Flt3L、VEGF、IL2、IL3、IL6和IL15的生长因子和细胞因子；
    其中iNK-B包含一种或多种选自SCF、Flt3L、IGF、IL7、IL2、IL3、IL6和IL15的生长因子和细胞因子。
  37. 如权利要求36所述的组合物，其中，
    GSK3β抑制剂是CHIR99021、BIO和/或肯帕罗酮；或
    VEGF通路激活剂是Deoxyshikonin、Isoarnebin I和/或Isoarnebin4；或
    cAMP通路激活剂是Forskolin和/或PACAP 1-38；或
    Notch信号通路激活剂是Resveratrol和/或VPA；或
    TGFβ受体/ALK抑制剂是SB431542、E-616452和/或A83-01；或
    所述造血干细胞促增殖分子是UM729、UM171和/或SR1。
  38. 权利要求36的组合物，其中，
    iHSC-B不含GSK3β抑制剂、Activin A和/或bFGF；
    iHSC-C不含GSK3β抑制剂、VEGF通路激活剂、cAMP通路激活剂和/或BMP激活剂；或
    iHSC-D不含bFGF、GSK3β抑制剂和/或BMP激活剂。
  39. 化合物A用于使多能干细胞诱导分化成中胚层的用途，其中，化合物A为
    其中，R1选自：
    R2为
    其中，X4、X5、X6、或X7选自H、甲基、乙基、羟基、甲氧基、乙氧基、或卤素，X4、X5、X6、或X7可以相同，也可以不同；
    优选R2选自以下任一结构：
    优选所述化合物A为
    所述中胚层细胞能够提供造血细胞谱系，所述造血细胞谱系包括：造血内皮、造血干细胞和造血祖细胞、造血多能祖细胞、T祖细胞、NK祖细胞、T细胞、NK细胞、NKT细胞和/或B细胞；且在中胚层诱导阶段不形成拟胚体。
  40. Deoxyshikonin、Isoarnebin I和/或Isoarnebin 4用于将多能干细胞来源的中胚层细胞分化成造血内皮的用途。
  41. Deoxyshikonin、Isoarnebin I、Isoarnebin 4和/或Resveratrol用于将多能干细胞来源的中胚层细胞分化成造血干细胞的用途。
  42. Resveratrol用于将多能干细胞来源的造血内皮分化成造血干细胞的用途。
PCT/CN2023/073316 2022-01-20 2023-01-20 诱导细胞分化的方法 WO2023138680A1 (zh)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN202210066079.2 2022-01-20
CN202210066079 2022-01-20
CN202210935441.5 2022-08-03
CN202210935441 2022-08-03
CN202210932654 2022-08-04
CN202210932654.2 2022-08-04

Publications (3)

Publication Number Publication Date
WO2023138680A1 true WO2023138680A1 (zh) 2023-07-27
WO2023138680A9 WO2023138680A9 (zh) 2023-08-31
WO2023138680A8 WO2023138680A8 (zh) 2023-11-02

Family

ID=87347876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/073316 WO2023138680A1 (zh) 2022-01-20 2023-01-20 诱导细胞分化的方法

Country Status (1)

Country Link
WO (1) WO2023138680A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180072992A1 (en) * 2015-01-26 2018-03-15 Fate Therapeutics, Inc. Methods and compositions for inducing hematopoietic cell differentiation
US20180320137A1 (en) * 2015-11-04 2018-11-08 Fate Therapeutics, Inc. Methods and compositions for inducing hematopoietic cell differentiation
CN113462642A (zh) * 2021-08-12 2021-10-01 呈诺再生医学科技(珠海横琴新区)有限公司 间充质干细胞的快速诱导分化方法、试剂盒及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180072992A1 (en) * 2015-01-26 2018-03-15 Fate Therapeutics, Inc. Methods and compositions for inducing hematopoietic cell differentiation
US20180320137A1 (en) * 2015-11-04 2018-11-08 Fate Therapeutics, Inc. Methods and compositions for inducing hematopoietic cell differentiation
CN113462642A (zh) * 2021-08-12 2021-10-01 呈诺再生医学科技(珠海横琴新区)有限公司 间充质干细胞的快速诱导分化方法、试剂盒及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU GUANG-HUI, MA QING-WEN;ZENG FAN-YI: "Mesoderm formation and hematopoietic lineage commitment from mesoderm", CHINESE BULLETIN OF LIFE SCIENCES, vol. 31, no. 11, 28 November 2019 (2019-11-28), pages 1116 - 1125, XP093079474, ISSN: 1004-0374, DOI: 10.13376/j.cbls/2019137 *
YODA KIYOMI; OHNUKI YOSHITSUGU; MASUI SHINJI; KUROSAWA HIROSHI: "Optimized conditions for the supplementation of human-induced pluripotent stem cell cultures with a GSK-3 inhibitor during embryoid body formation with the aim of inducing differentiation into mesodermal and cardiac lineage", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 129, no. 3, 12 October 2019 (2019-10-12), NL , pages 371 - 378, XP086068216, ISSN: 1389-1723, DOI: 10.1016/j.jbiosc.2019.09.015 *

Also Published As

Publication number Publication date
WO2023138680A9 (zh) 2023-08-31
WO2023138680A8 (zh) 2023-11-02

Similar Documents

Publication Publication Date Title
JP7236476B2 (ja) 造血細胞分化を誘導するための方法および組成物
US11162076B2 (en) Methods and compositions for inducing hematopoietic cell differentiation
CN112041428B (zh) 用于在悬浮培养物中分化人多能干细胞系的方法
CN114269906A (zh) 从多能干细胞产生造血祖细胞的方法
CN114341349A (zh) 造血诱导培养基
AU2020334258A1 (en) T cell production from RAG inactivated IPSCs
US20220267729A1 (en) Methods of t cell production
WO2023138680A1 (zh) 诱导细胞分化的方法
JP7072756B2 (ja) 多能性幹細胞から中胚葉前駆細胞および血液血管前駆細胞への分化誘導法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23742995

Country of ref document: EP

Kind code of ref document: A1